Chemically encoded spatially addressed library screening platforms

ABSTRACT

Provided herein are encoded split pool libraries useful, inter alia, for forming highly diverse and dense arrays for screening and detection of a variety of molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/555,531, filed Aug. 29, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/553,140, filed Aug. 23, 2017, which is anational stage under 35 U.S.C. § 371 of International Application No.PCT/US2016/019426, filed Feb. 24, 2016, wherein InternationalApplication No. PCT/US2016/019426 claims the benefit of U.S. ProvisionalApplication No. 62/120,262, filed Feb. 24, 2015, the contents of each ofwhich are herein incorporated by reference in their entireties and forall purposes.

INCORPORATION OF SEQUENCE LISTING

A copy of the Sequence Listing written in file Seq_Listing_P34713US03,created Jul. 29, 2020, 6,101 bytes, machine format IBM-PC, MS Windowsoperating system, is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The number of molecules displayed by arrays used for screening anddetection methods is restricted by the synthetic method of the array. Intheory, split pool synthesis can generate enormous libraries—limitedonly by the number of chemical steps and number of unique buildingblocks utilized per step (i.e. a 5 step library utilizing 100 uniquebuilding blocks per step would in theory yield a 100⁵ or 10 billionmember chemical library). However, in practice, encoded split poolstrategies face numerous practical constraints. Libraries that aredecodable but not screenable or vice versa are not useful. The encodingstrategy may be practically limited in the number and type of chemicalsteps or building blocks used. An encoded split pool library platformwhich requires large particles for decoding (e.g., by radio frequencytags or mass spectrometry) will normally need to contain fewer librarymembers than a similar library that can be created on smaller particles.If assays are to be performed on a particle, the ligand density on eachparticle and the surface chemistry environment around each ligand shouldnot interfere with the assay. The serial nature of reported decodingstrategies also limits the number of “hits” which can be identified in acost effective manner in a given screen, and therefore can limit thesize of a library that is screened. The present invention addressesthese and other problems in the art.

BRIEF SUMMARY OF THE INVENTION

In one aspect, a microparticle is provided. The microparticle iscovalently attached to a ligand domain through a first linker; and anucleic acid domain through a second linker, wherein the second linkeris cleavable and the first linker is not cleavable under a conditionthat the second linker is cleavable.

In another aspect, a solid support attached to a microparticle isprovided, wherein the microparticle is covalently attached to (i) aligand domain through a first linker; and (ii) a cleaved linker moiety.

In another aspect, a method of forming a cleaved microparticle isprovided. The method includes attaching a microparticle as providedherein including embodiments thereof to a solid support, thereby formingan immobilized microparticle. The second linker of the immobilizedmicroparticle is cleaved, thereby forming a cleaved microparticle.

In another aspect, a method of detecting a ligand binder is provided.The method includes (i) attaching a microparticle as provided hereinincluding embodiments thereof to a solid support, thereby forming animmobilized microparticle. (ii) A complementary nucleic acid is bound tothe nucleic acid domain of the immobilized microparticle and a locationof the nucleic acid domain on the solid support is determined, therebyforming a decoded and mapped microparticle. (iii) The second linker ofthe decoded and mapped microparticle is cleaved, thereby forming amapped and cleaved microparticle. (vi) A ligand binder is bound to theligand domain of the mapped and cleaved microparticle; and (v) alocation of the bound ligand binder on the solid support is identified,thereby detecting the ligand binder.

In another aspect, a method of detecting a ligand binder is provided.The method includes (i) contacting a ligand binder with a microparticleas provided herein including embodiments thereof thereby forming a boundligand binder. (ii) A location of the bound ligand binder is identifiedon the solid support, thereby detecting the ligand binder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic illustrating general stucture of microparticles usedto create library. Variation in ratios of A, B, and C are achievedthrough one or more synthetic transformations applied to the startingmaterial ProMag 1 COOH series particles. Initial target ratios are setby relative ratios of mixtures of building blocks used to assemble theparticle surface. The final ratios achieved are measured through acombination of TGA, LC/MS or gel analysis of cleaved products, andcolorimetric or fluorescence based solid phase assays. Preferred ranges:X has a concentration of 0.1 to 100 nanomoles/mg; 1% X≤A≤20% X; 40%X≤B≤99% X; 0% X≤C≤50% X; wherein X is A (library molecule attachmentpoint), B (encoding tag attachment point) or C (core immobilizationpoint).

FIG. 2: General schematic for how encoded, split pool synthesis can beperformed; emphasizing the exponential increase in chemical diversityobserved with a linear increase in the number of chemical steps andbuilding blocks. For a general review, see: Czarnik, A. W. “Encodingmethods for combinatorial chemistry,” Curr. Opin. Chem. Biol. 1997, 1,60-66. Example: 100 building blocks, 6 rounds of split-pool, 1×10¹²unique members, 600 chemical reactions. n is the number of splitpoolrounds, x is the number of building blocks, unique librarymemebers=X^(n), chemical reactions=n^(X)

FIG. 3: An illustration of the chemical bonds being formed and brokenduring one round of an encoded synthesis step using peptide synthesis asthe chemistry which is being encoded. In step 1 an Fmoc protected aminoacid is coupled to the free amine on the microparticle through an amidecoupling. In step 2 a nucleic acid tag bearing an alkyne and freeprimary amine is coupled to the azide group on the microparticle througha copper catalyzed Huisgen cycloaddition. In step 3, an azide group iscoupled to the primary amine on the nucleic acid through an amidecoupling. In step 4 the Fmoc group protecting the amine group of theincorporated amino acid is removed. The product following step 4displays the same reactive moieties as the starting material (prior tostep 1), but contains an additional nucleic acid tag and an additionalamino acid. See materials and methods for reaction details. Seematerials and methods for a detailed example of how initialmicroparticles are prepared for the orthogonal synthesis.

FIG. 4: Schematic illustrating the connectivity of the nucleic acid tagsin one of the potential tagging approaches. In this variant, during eachtagging step, all of the tag attachment points on the microparticles areconsumed, and an equivalent amount of new tag attachment points are thencreated on the ends of the newly attached nucleic acid tags. Two taggingsteps in this example would lead to the generation of predominantly asingle population of nucleic acid containing oligomers on each beadconsisting of tag 1 linked to tag 2. Note that by design, each tagpopulation is independently decodable, such that decodability is onlydependent on the presence of each individual tag—i.e. decodability oftag 2 is not dependent on being directly attached to tag 1 and viceversa—such that subsets of beads which may have not gone to completeconversion in either tagging step are still decodable, as long assufficient amounts of each tag are present for decoding.

FIG. 5: Schematic illustrating the connectivity of the nucleic acid tagsin one of the potential tagging approaches. In this variant, during eachtagging step, only a fraction of the tag attachment points on themicroparticle for the tags are consumed. As shown, two tagging steps inthis example would lead to the generation two populations of nucleicacid containing oligomers on each bead-one population consisting of tag1 and one population consisting of tag 2. For four tagging steps, fourunique populations per bead would be generated.

FIG. 6: Schematic illustrating the connectivity of the nucleic acid tagsin one of the potential tagging approaches. In this variant, during eachtagging step, a fraction of the tag attachment points on themicroparticles are consumed, and an equivalent amount of new tagattachment points are then created on the ends of the newly attachednucleic acid tags. Two tagging steps in this example would lead to thegeneration of three populations of nucleic acid containing oligomers oneach bead, tag 1, tag 2, and an oligomer consisting of tag 1 linked totag 2. Note that by design, each tag population is independentlydecodable, such that decodability of each tag is only dependent on thepresence of each individual tag—ie decodability of tag 2 is notdependent on being directly attached to tag 1 and vice versa.

FIG. 7: LC traces of the different tag intermediates during one round oftagging and the addition of a new tag linking element. As demonstratedby changes in retention time and mass spectra, the solid phasecycloaddition chemistry yields predominantly the desired product.Following the solid phase amide coupling of a new tag linking element,LC/MS analysis indicates conversion to the desired product. Seematerials and methods for cycloaddition conditions, amide couplingconditions, and cleavage conditions.

FIG. 8: A portion of the PEG modified microparticles was functionalizedwith an acid labile linker and submitted to a seven step solid phasepeptide synthesis. The product was cleaved from the microparticles usingTFA. The TFA was removed and residue was analyzed by mass spectrometry,demonstrating successful synthesis of the desired peptide.

FIG. 9A-9B: Demonstration of two different modes of libraryimmobilization. In FIG. 9A, a brightfield image of a portion ofApplicants' microparticles covalently immobilized to an activatedcarboxymethyldextran coated slide through amide bond formation. In FIG.9B on right, an SEM image of a portion of Applicants' microparticlesnoncovalently immobilized within a custom microfabricated silicon wafer.

FIG. 10: Microparticles undergoing four rounds of encoding/tagging steps(as outlined in Encoding Scheme of FIG. 6 can be successfully decoded.Two different portions of microparticles were submitted in parallel tofour rounds of tagging as illustrated in FIG. 6. Each portion ofmicroparticles was tagged with a unique 18 mer at each tagging step.Following the four rounds of encoding, each portion was exposed to aseries of hybridization solutions containing a 1:1 mixture of twodifferent fluorescently labeled oligonucleotide probes. Shown are eightdifferent two-channel fluorescent images of aliquots of the taggedmicroparticles following hybridization. In column 1, the twomicroparticle samples were hybridized with a 1:1 mixture of Cy3 labeledoligonucleotide complementary to A0, and a FITC labeled oligonucleotidecomplementary to A1. As expected, those particles encoded with tag A0fluoresced in the red (Cy3) channel (top image, column 1) and thoseparticles encoded with tag A1 fluoresced in the green (FITC) channel(bottom image, column 2). The trend holds true for all of the otherhybridization conditions, in which particles selectively fluoresce inthe wavelength corresponding to the fluorescent label attached to thecomplementary DNA sequence tag, rather than a mismatched tag.

FIG. 11: Microparticles undergoing four rounds of encoding/tagging steps(as outlined in FIG. 5 can be successfully decoded. Two differentportions of microparticles were submitted in parallel to four rounds oftagging as illustrated in FIG. 5. Each portion of microparticles wastagged with a unique 18 mer at each tagging step. Following the fourrounds of encoding, each portion was exposed to a series ofhybridization solutions containing a 1:1 mixture of two differentfluorescently labeled oligonucleotide probes. Shown are eight differenttwo-channel fluorescent images of aliquots of the tagged microparticlesfollowing hybridization. In column 1, the two microparticle samples werehybridized with a 1:1 mixture of Cy3 labeled oligonucleotidecomplementary to A0, and a FITC labeled oligonucleotide complementary toA1. As expected, those particles encoded with tag A0 fluoresced in thered (Cy3) channel (top image, column 1) and those particles encoded withtag A1 fluoresced in the green (FITC) channel (bottom image, column 2).The trend holds true for all of the other hybridization conditions, inwhich particles selectively fluoresce in the wavelength corresponding tothe fluorescent label attached to the complementary DNA sequence tag,rather than a mismatched tag.

FIG. 12: To demonstrate peptide synthesis is compatible with Applicants'encoding chemistry, two peptides (HA and Myc—two common antibodyepitopes) were synthesized in parallel on the microparticles in whichfour of the synthetic steps were encoded with DNA tags. The particleswere incubated with a Cy5 labeled antibody specific to the HA epitope atvarious steps in the synthesis. Microparticles that displayed DNA tagsand fully protected HA peptide (in which the side chain protectingmoieties have not been removed) did not bind the labeled antibody (toprow images). Following cleavage of the DNA tags, the microparticles thatdisplay side chain protected HA peptide do not bind the labeled antibody(second row images). Following deprotection of the side chain protectingmoieties (TFA treatment), the HA displaying microparticles now bind thelabeled antibody (third row images). Microparticles displaying fullydeprotected Myc peptide does not bind the labeled antibody (fourth rowimages)

FIG. 13: To demonstrate peptide synthesis is compatible with Applicants'encoding chemistry, two peptides (HA and Myc—two common antibodyepitopes) were synthesized in parallel on microparticles in which fourof the synthetic steps were encoded with DNA tags. The particles wereincubated with an Alexafluor 488 labeled antibody specific to the Mycepitope at various steps in the synthesis. Microparticles that displayedDNA tags and fully protected Myc peptide (in which the side chainprotecting moieties have not been removed) did not bind the labeledantibody (top row images). Following cleavage of the DNA tags, themicroparticles that display side chain protected Myc peptide do not bindthe labeled antibody (second row images). Following deprotection of theside chain protecting moieties (TFA treatment), the Myc displayingmicroparticles now bind the labeled antibody (third row images).Microparticles displaying fully deprotected HA peptide does not bind thelabeled antibody (fourth row images)

FIG. 14: Mixing the beads and stains demonstrates ability todifferentiate binding. Tagless beads bearing fully deprotected HApeptide or Myc peptide were mixed and stained with a mixture ofAnti-HA-Cy5 and Anti-Myc-Alexa 488. Fluorescence imaging indicates twodistinct bead populations.

FIG. 15A-FIG. 15H: FIG. 15A shows SEM of the 0.88 μm Promag particles.FIG. 15B shows a Silicon chip with 1.3 μm center to center spacing ofwells in a hexagonal pattern. FIG. 15C shows a Silicon chip with 1.3 μmcenter to center spacing of wells in a square pattern. FIG. 15D shows aSilicon chip with 1.3 μm center to center spacing of wells in a squarepattern-partially filled with microspheres. FIG. 15E shows a Siliconchip with 1.3 μm center to center spacing of wells in a hexagonalpattern, filled with microspheres. FIG. 15F shows a Silicon chip with1.3 μm center to center spacing of wells in a hexagonal pattern, filledwith microspheres-tilted. FIG. 15G shows a fluorescent Image ofAlexafluor 488-labeled microspheres immobilized in partially filledsilicon chip with 1.3 μm center to center spacing of wells in ahexagonal pattern. FIG. 15H shows a bright field image of microspheresimmobilized in a partially filled quartz chip with 1.3 μm center tocenter spacing of wells in a hexagonal pattern.

FIG. 16A, FIG. 16B, FIG. 16AC, AND FIG. 16D: FIG. 16A showsautofluorescence of microspheres immobilized in a partially filledquartz chip with 1.3 μm center to center spacing of wells in a hexagonalpattern. FIG. 16B shows a Silicon chip with 2.4 μm center to centerspacing of wells in a hexagonal pattern. FIG. 16AC shows a Silicon chipwith 2.4 μm center to center spacing of wells in a hexagonal pattern,partially filled with microspheres. FIG. 16D shows a fluorescent imageof DNA tagged-microspheres hybridized with fluorescently labeled DNAcomplements immobilized in a silicon chip. The chip is partially filledand has 2.4 μm center to center spacing of wells in a hexagonal pattern.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

While various embodiments and aspects of the present invention are shownand described herein, it will be obvious to those skilled in the artthat such embodiments and aspects are provided by way of example only.Numerous variations, changes, and substitutions will now occur to thoseskilled in the art without departing from the invention. It should beunderstood that various alternatives to the embodiments of the inventiondescribed herein may be employed in practicing the invention.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in the applicationincluding, without limitation, patents, patent applications, articles,books, manuals, and treatises are hereby expressly incorporated byreference in their entirety for any purpose.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchednon-cyclic carbon chain (or carbon), or combination thereof, which maybe fully saturated, mono- or polyunsaturated and can include di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example,n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkylgroup is one having one or more double bonds or triple bonds. Examplesof unsaturated alkyl groups include, but are not limited to, vinyl,2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and thehigher homologs and isomers. An alkoxy is an alkyl attached to theremainder of the molecule via an oxygen linker (—O—). An alkyl moietymay be an alkenyl moiety. An alkyl moiety may be an alkynyl moiety. Analkyl moiety may be fully saturated.

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred in the presentinvention. A “lower alkyl” or “lower alkylene” is a shorter chain alkylor alkylene group, generally having eight or fewer carbon atoms. Theterm “alkenylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkene.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable non-cyclic straight or branchedchain, or combinations thereof, including at least one carbon atom andat least one heteroatom selected from the group consisting of O, N, P,Si, and S, and wherein the nitrogen and sulfur atoms may optionally beoxidized, and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) O, N, P, S, and Si may be placed at any interior positionof the heteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. Examples include, but are notlimited to: —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —C H₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and—CN. Up to two or three heteroatoms may be consecutive, such as, forexample, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. A heteroalkyl moiety mayinclude one heteroatom (e.g., O, N, S, Si, or P). A heteroalkyl moietymay include two optionally different heteroatoms (e.g., O, N, S, Si, orP). A heteroalkyl moiety may include three optionally differentheteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may includefour optionally different heteroatoms (e.g., O, N, S, Si, or P). Aheteroalkyl moiety may include five optionally different heteroatoms(e.g., O, N, S, Si, or P). A heteroalkyl moiety may include up to 8optionally different heteroatoms (e.g., O, N, S, Si, or P).

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—. As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, —NR′R″, —OR, —SR′, and/or —SO₂R′. Where “heteroalkyl”is recited, followed by recitations of specific heteroalkyl groups, suchas —NR′R″ or the like, it will be understood that the terms heteroalkyland —NR′R″ are not redundant or mutually exclusive. Rather, the specificheteroalkyl groups are recited to add clarity. Thus, the term“heteroalkyl” should not be interpreted herein as excluding specificheteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated,non-aromatic cyclic versions of “alkyl” and “heteroalkyl,” respectively,wherein the carbons making up the ring or rings do not necessarily needto be bonded to a hydrogen due to all carbon valencies participating inbonds with non-hydrogen atoms. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl,3-hydroxy-cyclobut-3-enyl-1,2, dione, 1H-1,2,4-triazolyl-5(4H)-one,4H-1,2,4-triazolyl, and the like. Examples of heterocycloalkyl include,but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl,tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A“cycloalkylene” and a “heterocycloalkylene,” alone or as part of anothersubstituent, means a divalent radical derived from a cycloalkyl andheterocycloalkyl, respectively. A heterocycloalkyl moiety may includeone ring heteroatom (e.g., O, N, S, Si, or P). A heterocycloalkyl moietymay include two optionally different ring heteroatoms (e.g., O, N, S,Si, or P). A heterocycloalkyl moiety may include three optionallydifferent ring heteroatoms (e.g., O, N, S, Si, or P). A heterocycloalkylmoiety may include four optionally different ring heteroatoms (e.g., O,N, S, Si, or P). A heterocycloalkyl moiety may include five optionallydifferent ring heteroatoms (e.g., O, N, S, Si, or P). A heterocycloalkylmoiety may include up to 8 optionally different ring heteroatoms (e.g.,O, N, S, Si, or P).

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. The term “heteroaryl” refers to aryl groups (or rings)that contain at least one heteroatom such as N, O, or S, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. Thus, the term “heteroaryl” includesfused ring heteroaryl groups (i.e., multiple rings fused togetherwherein at least one of the fused rings is a heteroaromatic ring). A5,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 5 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. Likewise, a 6,6-fused ringheteroarylene refers to two rings fused together, wherein one ring has 6members and the other ring has 6 members, and wherein at least one ringis a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to tworings fused together, wherein one ring has 6 members and the other ringhas 5 members, and wherein at least one ring is a heteroaryl ring. Aheteroaryl group can be attached to the remainder of the moleculethrough a carbon or heteroatom. Non-limiting examples of aryl andheteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl,1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl,4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl,5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl,4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl,2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl,5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and6-quinolyl. Substituents for each of the above noted aryl and heteroarylring systems are selected from the group of acceptable substituentsdescribed below. An “arylene” and a “heteroarylene,” alone or as part ofanother substituent, mean a divalent radical derived from an aryl andheteroaryl, respectively. Non-limiting examples of aryl and heteroarylgroups include pyridinyl, pyrimidinyl, thiophenyl, thienyl, furanyl,indolyl, benzoxadiazolyl, benzodioxolyl, benzodioxanyl, thianaphthanyl,pyrrolopyridinyl, indazolyl, quinolinyl, quinoxalinyl, pyridopyrazinyl,quinazolinonyl, benzoisoxazolyl, imidazopyridinyl, benzofuranyl,benzothienyl, benzothiophenyl, phenyl, naphthyl, biphenyl, pyrrolyl,pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl,furylthienyl, pyridyl, pyrimidyl, benzothiazolyl, purinyl,benzimidazolyl, isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl,diazolyl, triazolyl, tetrazolyl, benzothiadiazolyl, isothiazolyl,pyrazolopyrimidinyl, pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl,or quinolyl. The examples above may be substituted or unsubstituted anddivalent radicals of each heteroaryl example above are non-limitingexamples of heteroarylene. A heteroaryl moiety may include one ringheteroatom (e.g., O, N, or S). A heteroaryl moiety may include twooptionally different ring heteroatoms (e.g., O, N, or S). A heteroarylmoiety may include three optionally different ring heteroatoms (e.g., O,N, or S). A heteroaryl moiety may include four optionally different ringheteroatoms (e.g., O, N, or S). A heteroaryl moiety may include fiveoptionally different ring heteroatoms (e.g., O, N, or S). An aryl moietymay have a single ring. An aryl moiety may have two optionally differentrings. An aryl moiety may have three optionally different rings. An arylmoiety may have four optionally different rings. A heteroaryl moiety mayhave one ring. A heteroaryl moiety may have two optionally differentrings. A heteroaryl moiety may have three optionally different rings. Aheteroaryl moiety may have four optionally different rings. A heteroarylmoiety may have five optionally different rings.

A fused ring heterocyloalkyl-aryl is an aryl fused to aheterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is aheteroaryl fused to a heterocycloalkyl. A fused ringheterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl.A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkylfused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl,fused ring heterocycloalkyl-heteroaryl, fused ringheterocycloalkyl-cycloalkyl, or fused ringheterocycloalkyl-heterocycloalkyl may each independently beunsubstituted or substituted with one or more of the substitutentsdescribed herein.

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

The term “alkylsulfonyl,” as used herein, means a moiety having theformula —S(O₂)—R′, where R is a substituted or unsubstituted alkyl groupas defined above. R may have a specified number of carbons (e.g., “C₁-C₄alkylsulfonyl”).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,”, “cycloalkyl”,“heterocycloalkyl”, “aryl,” and “heteroaryl”) includes both substitutedand unsubstituted forms of the indicated radical. Preferred substituentsfor each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″)═NR′″, —S(O)R′,—S(O)₂R′, —S(O)₂N(R)(‘R″—NRSO₂R’), —CN, and —NO₂ in a number rangingfrom zero to (2m′+1), where m′ is the total number of carbon atoms insuch radical. R′, R″, R′″, and R″″ each preferably independently referto hydrogen, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl (e.g., aryl substituted with 1-3halogens), substituted or unsubstituted alkyl, alkoxy, or thioalkoxygroups, or arylalkyl groups. When a compound of the invention includesmore than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″, and R″″ group when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 4-, 5-, 6-, or 7-membered ring. For example, —NR′R″ includes, but isnot limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR, —NR′R″, —SR′, -halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, NR″C(O)₂R′, NRC(NR′R″)═NR′″, S(O)R′, —S(O)₂R′,—S(O)₂N(R′)(R″, —NRSO₂R′), —CN, —NO₂, —R, —N₃, —CH(Ph)₂,fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in a number ranging fromzero to the total number of open valences on the aromatic ring system;and where R′, R″, R′″, and R″″ are preferably independently selectedfrom hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″,and R″″ groups when more than one of these groups is present.

Where a moiety is substituted with an R substituent, the group may bereferred to as “R-substituted.” Where a moiety is R-substituted, themoiety is substituted with at least one R substituent and each Rsubstituent is optionally different. For example, where a moiety hereinis R^(1A)-substituted or unsubstituted alkyl, a plurality of R^(1A)substituents may be attached to the alkyl moiety wherein each R^(1A)substituent is optionally different. Where an R-substituted moiety issubstituted with a plurality R substituents, each of the R-substituentsmay be differentiated herein using a prime symbol (′) such as R′, R″,etc. For example, where a moiety is R^(1A)-substituted or unsubstitutedalkyl, and the moiety is substituted with a plurality of R^(1A)substituents, the plurality of R^(1A) substituents may be differentiatedas R^(1A)′, R^(1A)″, R^(1A)′″, etc. In some embodiments, the pluralityof R substituents is 3. In some embodiments, the plurality of Rsubstituents is 2.

In embodiments, a compound as described herein may include multipleinstances of R¹, R², R³, R⁴, R⁵, R⁶, R^(6A), R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹²,R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, and/or othersubstituents and variables. In such embodiments, each variable mayoptional be different and be appropriately labeled to distinguish eachgroup for greater clarity. For example, where each R^(6A) is different,they may be referred to, for example, as R^(6A.1), R^(6A.2), R^(6A.3),or R^(6A.4), respectively, wherein the definition of R^(6A) is assumedby R^(6A.1), R^(6A.2), R^(6A.3), and/or R^(6A.4). The variables usedwithin a definition of R¹, R², R³, R⁴, R⁵, R⁶, R^(6A), R⁷, R⁸, R⁹, R¹⁰,R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, and/orother variables that appear at multiple instances and are different maysimilarly be appropriately labeled to distinguish each group for greaterclarity.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′—(C″R″R′″)_(d)—, where variables s and d areindependently integers of from 0 to 3, and X′ is —O—, —NR—, —S—, —S(O)—,—S(O)₂—, or —S(O)₂NR′—. The substituents R, R′, R″, and R′″ arepreferably independently selected from hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,        —NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH,        —NHOH, —OCF₃, —OCHF₂, unsubstituted alkyl, unsubstituted        heteroalkyl, unsubstituted cycloalkyl, unsubstituted        heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl,        and    -   (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,        heteroaryl, substituted with at least one substituent selected        from:        -   (i) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,            —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,            —NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH,            —NHOH, —OCF₃, —OCHF₂, unsubstituted alkyl, unsubstituted            heteroalkyl, unsubstituted cycloalkyl, unsubstituted            heterocycloalkyl, unsubstituted aryl, unsubstituted            heteroaryl, and        -   (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,            heteroaryl, substituted with at least one substituent            selected from:            -   (a) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,                —NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,                —NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H,                —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, unsubstituted alkyl,                unsubstituted heteroalkyl, unsubstituted cycloalkyl,                unsubstituted heterocycloalkyl, unsubstituted aryl,                unsubstituted heteroaryl, and            -   (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,                aryl, heteroaryl, substituted with at least one                substituent selected from: oxo, halogen, —CF₃, —CN, —OH,                —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H,                —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O) NH₂,                —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂,                unsubstituted alkyl, unsubstituted heteroalkyl,                unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, unsubstituted                heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl.

In some embodiments, each substituted group described in the compoundsherein is substituted with at least one substituent group. Morespecifically, in some embodiments, each substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene described in the compounds herein are substituted with atleast one substituent group. In other embodiments, at least one or allof these groups are substituted with at least one size-limitedsubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted orunsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl. In someembodiments of the compounds herein, each substituted or unsubstitutedalkylene is a substituted or unsubstituted C₁-C₂₀ alkylene, eachsubstituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 20 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₈cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 8 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 10 membered heteroarylene.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and/or each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl. In some embodiments, each substituted orunsubstituted alkylene is a substituted or unsubstituted C₁-C₈ alkylene,each substituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 7 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 9 membered heteroarylene. In someembodiments, the compound is a chemical species set forth in theExamples section, figures, or tables below.

The terms “a” or “an,” as used in herein means one or more. In addition,the phrase “substituted with a[n],” as used herein, means the specifiedgroup may be substituted with one or more of any or all of the namedsubstituents. For example, where a group, such as an alkyl or heteroarylgroup, is “substituted with an unsubstituted C₁-C₂₀ alkyl, orunsubstituted 2 to 20 membered heteroalkyl,” the group may contain oneor more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2to 20 membered heteroalkyls. Moreover, where a moiety is substitutedwith an R substituent, the group may be referred to as “R-substituted.”Where a moiety is R-substituted, the moiety is substituted with at leastone R substituent and each R substituent is optionally different.

The symbol “

” denotes the point of attachment of a chemical moiety to the remainderof a molecule or chemical formula.

Descriptions of compounds of the present invention are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York,N.Y. 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL,Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). Any methods,devices and materials similar or equivalent to those described hereincan be used in the practice of this invention. The following definitionsare provided to facilitate understanding of certain terms usedfrequently herein and are not meant to limit the scope of the presentdisclosure.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single-, double- or multiple-stranded form,or complements thereof. The term “polynucleotide” refers to a linearsequence of nucleotides. The term “nucleotide” typically refers to asingle unit of a polynucleotide, i.e., a monomer. Nucleotides can beribonucleotides, deoxyribonucleotides, or modified versions thereof.Examples of polynucleotides contemplated herein include single anddouble stranded DNA, single and double stranded RNA (including siRNA),and hybrid molecules having mixtures of single and double stranded DNAand RNA. Nucleic acids can be linear or branched. For example, nucleicacids can be a linear chain of nucleotides or the nucleic acids can bebranched, e.g., such that the nucleic acids comprise one or more arms orbranches of nucleotides. Optionally, the branched nucleic acids arerepetitively branched to form higher ordered structures such asdendrimers and the like.

Nucleic acids, including nucleic acids with a phosphothioate backbonecan include one or more reactive moieties. As used herein, the termreactive moiety includes any group capable of reacting with anothermolecule, e.g., a nucleic acid or polypeptide through covalent,non-covalent or other interactions. By way of example, the nucleic acidcan include an amino acid reactive moiety that reacts with an amino acidon a protein or polypeptide through a covalent, non-covalent or otherinteraction.

The terms also encompass nucleic acids containing known nucleotideanalogs or modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, which have similarbinding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphodiester derivativesincluding, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate(also known as phosphothioate), phosphorodithioate, phosphonocarboxylicacids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformicacid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamiditelinkages (see Eckstein, Oligonucleotides and Analogues: A PracticalApproach, Oxford University Press); and peptide nucleic acid backbonesand linkages. Other analog nucleic acids include those with positivebackbones; non-ionic backbones, modified sugars, and non-ribosebackbones (e.g. phosphorodiamidate morpholino oligos or locked nucleicacids (LNA)), including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Sanghui & Cook, eds. Nucleic acidscontaining one or more carbocyclic sugars are also included within onedefinition of nucleic acids. Modifications of the ribose-phosphatebackbone may be done for a variety of reasons, e.g., to increase thestability and half-life of such molecules in physiological environmentsor as probes on a biochip. Mixtures of naturally occurring nucleic acidsand analogs can be made; alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made. In embodiments, the internucleotide linkages in DNAare phosphodiester, phosphodiester derivatives, or a combination ofboth.

Nucleic acids can include nonspecific sequences. As used herein, theterm “nonspecific sequence” refers to a nucleic acid sequence thatcontains a series of residues that are not designed to be complementaryto or are only partially complementary to any other nucleic acidsequence. By way of example, a nonspecific nucleic acid sequence is asequence of nucleic acid residues that does not function as aninhibitory nucleic acid when contacted with a cell or organism. An“inhibitory nucleic acid” is a nucleic acid (e.g. DNA, RNA, polymer ofnucleotide analogs) that is capable of binding to a target nucleic acid(e.g. an mRNA translatable into a protein) and reducing transcription ofthe target nucleic acid (e.g. mRNA from DNA) or reducing the translationof the target nucleic acid (e.g. mRNA) or altering transcript splicing(e.g. single stranded morpholino oligo).

A “labeled nucleic acid or oligonucleotide” is one that is bound, eithercovalently, through a linker or a chemical bond, or noncovalently,through ionic, van der Waals, electrostatic, or hydrogen bonds to alabel such that the presence of the nucleic acid may be detected bydetecting the presence of the detectable label bound to the nucleicacid. Alternatively, a method using high affinity interactions mayachieve the same results where one of a pair of binding partners bindsto the other, e.g., biotin, streptavidin. In embodiments, the nucleicacid domain includes a detectable label, as disclosed herein andgenerally known in the art.

The term “probe” or “primer”, as used herein, is defined to be one ormore nucleic acid fragments whose specific hybridization to a sample canbe detected. A probe or primer can be of any length depending on theparticular technique it will be used for. For example, PCR primers aregenerally between 10 and 40 nucleotides in length, while nucleic acidprobes for, e.g., a Southern blot, can be more than a hundrednucleotides in length. The probe may be unlabeled or labeled asdescribed below so that its binding to the target or sample can bedetected. The probe can be produced from a source of nucleic acids fromone or more particular (preselected) portions of a chromosome, e.g., oneor more clones, an isolated whole chromosome or chromosome fragment, ora collection of polymerase chain reaction (PCR) amplification products.The length and complexity of the nucleic acid fixed onto the targetelement is not critical to the invention. One of skill can adjust thesefactors to provide optimum hybridization and signal production for agiven hybridization procedure, and to provide the required resolutionamong different genes or genomic locations.

The probe may also be isolated nucleic acids immobilized on a solidsurface (e.g., nitrocellulose, glass, quartz, fused silica slides), asin an array. In some embodiments, the probe may be a member of an arrayof nucleic acids as described, for instance, in WO 96/17958. Techniquescapable of producing high density arrays can also be used for thispurpose (see, e.g., Fodor (1991) Science 767-773; Johnston (1998) Curr.Biol. 8: R171-R174; Schummer (1997) Biotechniques 23: 1087-1092; Kern(1997) Biotechniques 23: 120-124; U.S. Pat. No. 5,143,854).

The words “complementary” or “complementarity” refer to the ability of anucleic acid in a polynucleotide to form a base pair with anothernucleic acid in a second polynucleotide. For example, the sequence A-G-Tis complementary to the sequence T-C-A. Complementarity may be partial,in which only some of the nucleic acids match according to base pairing,or complete, where all the nucleic acids match according to basepairing.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apre-sequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a pre-protein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are near each other, and, inthe case of a secretory leader, contiguous and in reading phase.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “gene” means the segment of DNA involved in producing aprotein; it includes regions preceding and following the coding region(leader and trailer) as well as intervening sequences (introns) betweenindividual coding segments (exons). The leader, the trailer as well asthe introns include regulatory elements that are necessary during thetranscription and the translation of a gene. Further, a “protein geneproduct” is a protein expressed from a particular gene.

The word “expression” or “expressed” as used herein in reference to agene means the transcriptional and/or translational product of thatgene. The level of expression of a DNA molecule in a cell may bedetermined on the basis of either the amount of corresponding mRNA thatis present within the cell or the amount of protein encoded by that DNAproduced by the cell. The level of expression of non-coding nucleic acidmolecules (e.g., siRNA) may be detected by standard PCR or Northern blotmethods well known in the art. See, Sambrook et al., 1989 MolecularCloning: A Laboratory Manual, 18.1-18.88.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all. Transgenic cells and plants are thosethat express a heterologous gene or coding sequence, typically as aresult of recombinant methods.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion

The term “exogenous” refers to a molecule or substance (e.g., acompound, nucleic acid or protein) that originates from outside a givencell or organism. For example, an “exogenous promoter” as referred toherein is a promoter that does not originate from the plant it isexpressed by. Conversely, the term “endogenous” or “endogenous promoter”refers to a molecule or substance that is native to, or originateswithin, a given cell or organism.

The term “isolated”, when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It can be,for example, in a homogeneous state and may be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified.

The term “purified” denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. In some embodiments, thenucleic acid or protein is at least 50% pure, optionally at least 65%pure, optionally at least 75% pure, optionally at least 85% pure,optionally at least 95% pure, and optionally at least 99% pure.

The term “isolated” may also refer to a cell or sample cells. Anisolated cell or sample cells are a single cell type that issubstantially free of many of the components which normally accompanythe cells when they are in their native state or when they are initiallyremoved from their native state. In certain embodiments, an isolatedcell sample retains those components from its natural state that arerequired to maintain the cell in a desired state. In some embodiments,an isolated (e.g. purified, separated) cell or isolated cells, are cellsthat are substantially the only cell type in a sample. A purified cellsample may contain at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% of one type of cell. An isolated cell sample may beobtained through the use of a cell marker or a combination of cellmarkers, either of which is unique to one cell type in an unpurifiedcell sample. In some embodiments, the cells are isolated through the useof a cell sorter. In some embodiments, antibodies against cell proteinsare used to isolate cells.

As used herein, the term “conjugate” refers to the association betweenatoms or molecules. The association can be direct or indirect. Forexample, a conjugate between a nucleic acid and a protein can be direct,e.g., by covalent bond, or indirect, e.g., by non-covalent bond (e.g.electrostatic interactions (e.g. ionic bond, hydrogen bond, halogenbond), van der Waals interactions (e.g. dipole-dipole, dipole-induceddipole, London dispersion), ring stacking (pi effects), hydrophobicinteractions and the like). In embodiments, conjugates are formed usingconjugate chemistry including, but are not limited to nucleophilicsubstitutions (e.g., reactions of amines and alcohols with acyl halides,active esters), electrophilic substitutions (e.g., enamine reactions)and additions to carbon-carbon and carbon-heteroatom multiple bonds(e.g., Michael reaction, Diels-Alder addition). These and other usefulreactions are discussed in, for example, March, ADVANCED ORGANICCHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney etal., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington, D.C., 1982. In embodiments, themicroparticle is non-covalently attached to a solid support through anon-covalent chemical reaction between a component of the microparticleand a component of the solid support. In other embodiments, themicroparticle includes one or more reactive moieties, e.g., a covalentreactive moiety, as described herein (e.g., an amine reactive moiety).In other embodiments, the microparticle includes a linker with one ormore reactive moieties, e.g., a covalent reactive moiety, as describedherein (e.g., an amine reactive moiety).

Useful reactive moieties or reactive functional groups used forconjugate chemistries herein include, for example:

(a) carboxyl groups and various derivatives thereof including, but notlimited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters,acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl,alkenyl, alkynyl and aromatic esters;

(b) hydroxyl groups which can be converted to esters, ethers, aldehydes,etc.

(c) haloalkyl groups wherein the halide can be later displaced with anucleophilic group such as, for example, an amine, a carboxylate anion,thiol anion, carbanion, or an alkoxide ion, thereby resulting in thecovalent attachment of a new group at the site of the halogen atom;

(d) dienophile groups which are capable of participating in Diels-Alderreactions such as, for example, maleimido groups;

(e) aldehyde or ketone groups such that subsequent derivatization ispossible via formation of carbonyl derivatives such as, for example,imines, hydrazones, semicarbazones or oximes, or via such mechanisms asGrignard addition or alkyllithium addition;

(f) sulfonyl halide groups for subsequent reaction with amines, forexample, to form sulfonamides;

(g) thiol groups, which can be converted to disulfides, reacted withacyl halides, or bonded to metals such as gold;

(h) amine or sulfhydryl groups, which can be, for example, acylated,alkylated or oxidized;

(i) alkenes, which can undergo, for example, cycloadditions, acylation,Michael addition, etc;

(j) epoxides, which can react with, for example, amines and hydroxylcompounds;

(k) phosphoramidites and other standard reactive moieties useful innucleic acid synthesis;

(l) metal silicon oxide bonding;

(m) metal bonding to reactive phosphorus groups (e.g. phosphines) toform, for example, phosphate diester bonds; and

(n) sulfones, for example, vinyl sulfone.

The reactive moieties can be chosen such that they do not participatein, or interfere with, the chemical stability of the proteins or nucleicacids described herein. By way of example, the nucleic acids can includea vinyl sulfone or other reactive moiety (e.g., maleimide). Optionally,the nucleic acids can include a reactive moiety having the formulaS—S—R. R can be, for example, a protecting moiety. Optionally, R ishexanol. As used herein, the term hexanol includes compounds with theformula C₆H₁₃OH and includes, 1-hexanol, 2-hexanol, 3-hexanol,2-methyl-1-pentanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol,2-methyl-2-pentanol, 3-methyl-2-pentanol, 4-methyl-2-pentanol,2-methyl-3-pentanol, 3-methyl-3-pentanol, 2,2-dimethyl-1-butanol,2,3-dimethyl-1-butanol, 3,3-dimethyl-1-butanol, 2,3-dimethyl-2-butanol,3,3-dimethyl-2-butanol, and 2-ethyl-1-butanol. Optionally, R is1-hexanol.

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments, theterm “about” means within a standard deviation using measurementsgenerally acceptable in the art. In embodiments, about means a rangeextending to +/−10% of the specified value. In embodiments, about meansthe specified value.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues,wherein the polymer may be conjugated to a moiety that does not consistof amino acids. The terms apply to amino acid polymers in which one ormore amino acid residue is an artificial chemical mimetic of acorresponding naturally occurring amino acid, as well as to naturallyoccurring amino acid polymers and non-naturally occurring amino acidpolymers. The terms apply to macrocyclic peptides, peptides that havebeen modified with non-peptide functionality, peptidomimetics,polyamides, and macrolactams. A “fusion protein” refers to a chimericprotein encoding two or more separate protein sequences that arerecombinantly expressed as a single moiety.

The term “peptidyl” and “peptidyl moiety” means a monovalent peptide.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. The terms“non-naturally occurring amino acid” and “unnatural amino acid” refer toamino acid analogs, synthetic amino acids, and amino acid mimetics whichare not found in nature.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,or 99% identity over a specified region, e.g., of the entire polypeptidesequences of the invention or individual domains of the polypeptides ofthe invention), when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using one ofthe following sequence comparison algorithms or by manual alignment andvisual inspection. Such sequences are then said to be “substantiallyidentical.” This definition also refers to the complement of a testsequence. Optionally, the identity exists over a region that is at leastabout 50 nucleotides in length, or more preferably over a region that is100 to 500 or 1000 or more nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of, e.g., a full length sequence or from 20 to 600, about 50to about 200, or about 100 to about 150 amino acids or nucleotides inwhich a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned. Methods of alignment of sequences for comparison are well knownin the art. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith and Waterman(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci.USA 85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., CurrentProtocols in Molecular Biology (1995 supplement)).

An example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always>0) and N (penalty score for mismatchingresidues; always<0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross-reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.chemical compounds including biomolecules or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated; however, the resulting reaction product can be produceddirectly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture.

The term “contacting” may include allowing two species to react,interact, or physically touch, wherein the two species may be, forexample, a ligand domain as described herein and a ligand binder. Inembodiments contacting includes, for example, allowing a ligand domainas described herein to interact with a ligand binder.

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample.For example, a test sample can be taken from a test condition, e.g., inthe presence of a test compound, and compared to samples from knownconditions, e.g., in the absence of the test compound (negativecontrol), or in the presence of a known compound (positive control). Acontrol can also represent an average value gathered from a number oftests or results. One of skill in the art will recognize that controlscan be designed for assessment of any number of parameters. For example,a control can be devised to compare therapeutic benefit based onpharmacological data (e.g., half-life) or therapeutic measures (e.g.,comparison of side effects). One of skill in the art will understandwhich standard controls are most appropriate in a given situation and beable to analyze data based on comparisons to standard control values.Standard controls are also valuable for determining the significance(e.g. statistical significance) of data. For example, if values for agiven parameter are widely variant in standard controls, variation intest samples will not be considered as significant.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins or otherentities which can be made detectable, e.g., by incorporating aradiolabel into a peptide or antibody specifically reactive with atarget peptide. Any appropriate method known in the art for conjugatingan antibody to the label may be employed, e.g., using methods describedin Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., SanDiego.

A “labeled protein or polypeptide” is one that is bound, eithercovalently, through a linker or a chemical bond, or noncovalently,through ionic, van der Waals, electrostatic, or hydrogen bonds to alabel such that the presence of the labeled protein or polypeptide maybe detected by detecting the presence of the label bound to the labeledprotein or polypeptide. Alternatively, methods using high affinityinteractions may achieve the same results where one of a pair of bindingpartners binds to the other, e.g., biotin, streptavidin.

“Biological sample” or “sample” refer to materials obtained from orderived from a subject or patient. A biological sample includes sectionsof tissues such as biopsy and autopsy samples, and frozen sections takenfor histological purposes. Such samples include bodily fluids such asblood and blood fractions or products (e.g., serum, plasma, platelets,red blood cells, and the like), sputum, tissue, cultured cells (e.g.,primary cultures, explants, and transformed cells) stool, urine,synovial fluid, joint tissue, synovial tissue, synoviocytes,fibroblast-like synoviocytes, macrophage-like synoviocytes, immunecells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. Abiological sample is typically obtained from a eukaryotic organism, suchas a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat;a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; orfish.

A “cell” as used herein, refers to a cell carrying out metabolic orother function sufficient to preserve or replicate its genomic DNA. Acell can be identified by well-known methods in the art including, forexample, presence of an intact membrane, staining by a particular dye,ability to produce progeny or, in the case of a gamete, ability tocombine with a second gamete to produce a viable offspring. Cells mayinclude prokaryotic and eukaroytic cells. Prokaryotic cells include butare not limited to bacteria. Eukaryotic cells include but are notlimited to yeast cells and cells derived from plants and animals, forexample mammalian, insect (e.g., spodoptera) and human cells.

The term “antibody” is used according to its commonly known meaning inthe art. Antibodies exist, e.g., as intact immunoglobulins or as anumber of well-characterized fragments produced by digestion withvarious peptidases. Thus, for example, pepsin digests an antibody belowthe disulfide linkages in the hinge region to produce F(ab)′₂, a dimerof Fab which itself is a light chain joined to V_(H)-C_(H1) by adisulfide bond. The F(ab)′₂ may be reduced under mild conditions tobreak the disulfide linkage in the hinge region, thereby converting theF(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer is essentially Fabwith part of the hinge region (see Fundamental Immunology (Paul ed., 3ded. 1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole etal., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)).“Monoclonal” antibodies (mAb) refer to antibodies derived from a singleclone. Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptidesof this invention. Also, transgenic mice, or other organisms such asother mammals, may be used to express humanized antibodies.Alternatively, phage display technology can be used to identifyantibodies and heteromeric Fab fragments that specifically bind toselected antigens (see, e.g., McCafferty et al., Nature 348:552-554(1990); Marks et al., Biotechnology 10:779-783 (1992)).

A “solid support” as provided herein refers to any material that can bemodified to contain discrete individual sites appropriate for theattachment or association of a microparticle as provided hereinincluding embodiments thereof and is amenable to the methods providedherein including embodiments thereof. Examples of solid supports includewithout limitation, glass and modified or functionalized glass (e.g.,carboxymethyldextran functionalized glass), plastics (includingacrylics, polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™,etc.), polysaccharides, nylon or nitrocellulose, composite materials,ceramics, and plastic resins, silica or silica-based materials includingsilicon and modified silicon (e.g., patterned silicon), carbon, metals,quartz (e.g., patterned quartz), inorganic glasses, plastics, opticalfiber bundles, and a variety of other polymers. In general, thesubstrates allow optical detection and do not appreciably fluoresce.

The solid support provided herein including embodiments thereof may formpart of an ion-sensitive field-effect transistor (ISFET) microarray. Thesolid support may be planar (e.g., flat planar substrates such as glass,polystyrene and other plastics and acrylics). Although it will beappreciated by a person of ordinary skill in the art that otherconfigurations of solid supports may be used as well, for example, threedimensional configurations can be used. The solid support may bemodified to contain discrete, individual sites (also referred to hereinas “wells”) for microparticle binding. These sites generally includephysically altered sites, i.e. physical configurations such as wells orsmall depressions in the substrate that can retain the microparticles.The wells may be formed using a variety of techniques well known in theart, including, but not limited to, photolithography, stampingtechniques, molding techniques and microetching techniques. It will beappreciated by a person of ordinary skill in the art that the techniqueused will depend on the composition and shape of the solid support. Inembodiments, physical alterations are made in a surface of the solidsupport to produce wells. The required depth of the wells will depend onthe size of the microparticle to be added to the well.

A “microparticle” as used herein refers to a non-planar (e.g. spherical)particle having a size sufficient to attach molecules (e.g., a first, asecond or a third linker provided, a ligand domain and a nucleic aciddomain), directly or indirectly, through either covalent or non-covalentbonds. The microparticle may include any material that is capable ofproviding physical support for the molecules (e.g., a first, a second ora third linker provided, a ligand domain and a nucleic acid domain) thatare attached to the surface. The material is generally capable ofenduring conditions related to the attachment of the molecules (e.g., afirst, a second or a third linker provided, a ligand domain and anucleic acid domain) to the surface and any subsequent treatment,handling, or processing encountered during the performance of an assay.The materials may be naturally occurring, synthetic, or a modificationof a naturally occurring material. Suitable microparticle materials mayinclude silicon, ceramics, plastics (including polymers such as, e.g.,poly(vinyl chloride), cyclo-olefin copolymers, agarose, polyacrylamide,polyacrylate, polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate),polytetrafluoroethylene (PTFE or Teflon®), nylon, poly(vinyl butyrate)),germanium, gallium arsenide, gold or silver, copper or aluminumsurfaces, magnetic surfaces, e.g. Fe, Mn, Ni, Co, and their oxides,quantum dots, e.g., III-V (GaN, GaP, GaAs, InP, or InAs) or II-VI (ZnO,ZnS, CdS, CdSe, or CdTe) semiconductors, or Ln-doped fluoridenanocrystals, rare earth-doped oxidic nanomaterials either used bythemselves or in conjunction with other materials. Additional rigidmaterials may be considered, such as glass, which includes silica andfurther includes, for example, glass that is available as Bioglass.Other materials that may be employed include porous materials, such as,for example, controlled pore glass beads, crosslinked beaded Sepharose®or agarose resins, or copolymers of crosslinked bis-acrylamide andazalactone. Other beads include polymer beads, solid core beads,paramagnetic beads, or microbeads. Any other materials known in the artthat are capable of having one or more moieties, such as any of anamino, carboxyl, thiol, or hydroxyl reactive moiety, for example,incorporated on its surface, are also contemplated. In embodiments, themicroparticle is a magnetic polymer-based sphere. In embodiments, themicroparticle is a ProMag™ microsphere. In embodiments, the longestdimension of the microparticle is less than 1000 μm.

Compositions

The compositions provided herein are, inter alia, useful for theassembly of highly dense arrays suitable for a variety of highthroughput screening methods. The microparticles provided herein includea ligand domain attached through a first linker and a nucleic aciddomain attached through a second linker. By binding to a solid supportthe microparticles provided herein including embodiments thereof mayform part of an array. The ligand domain and the nucleic acid domain aresynthesized on the microparticle using methods of encoded split poolchemistry. Encoded split pool chemistry is a method well known in theart and described, inter alia, by the following references: Furka, A.;et al. Int. J. Pept. Protein Res. 1991, 37, 487-493; Kit Lam et al.,Nature, 1991; 354: 82-84; U.S. Pat. Nos. 6,060,596; 5,770,358;6,368,874; 5,565,324; 6,936,477 and 5,573,905; which are allincorporated by reference herein in their entirety and for all purposes.Each step of the ligand domain synthesis (e.g., peptide or chemicalcompound synthesis) is encoded in the nucleic acid domain by a shortnucleic acid sequence serving as an identification bar code. Therefore,each microparticle includes a unique ligand domain and a correspondingnucleic acid domain encoding specific nucleic acid sequences. Thespecific nucleic acid sequences correspond to the building blocks of theligand domain and the order in which they were incorporated in theligand domain Upon hybridization of a complementary nucleic acid to saidnucleic acid domain, the composition of the ligand domain and itslocation on the array can be determined (decoded). After the identity ofthe ligand domain and its location on the array have been determined,the nucleic acid domain is removed, the ligand domain may be furthermodified and contacted with a ligand binder (e.g., biomolecule).

In one aspect, a microparticle is provided. The microparticle iscovalently attached to a ligand domain through a first linker; and anucleic acid domain through a second linker, wherein the second linkeris cleavable and the first linker is not cleavable under a conditionthat the second linker is cleavable.

In embodiments, the microparticle is a microbead. A “microbead” asreferred to herein is a polymer-based microparticle of roughly sphericalshape with a diameter of about 0.5 μm to about 500 μm. The term“polymer-based” or “polymeric” as provided herein refers to amicroparticle or microbead including at least one polymer compound(e.g., polyethylene glycols, polyethylene imides, polysaccharides,polypeptides, or polynucleotides). In embodiments, the microbead is aProMag™ microsphere. In embodiments, the microbead is a polymer-basedmagnetic microbead.

The microparticle provided herein including embodiments thereof may beless than 200 μm. Where the microparticle is less than 200 μm a personof ordinary skill in the art will immediately recognize that the longestdimension (e.g. diameter or length) of a microparticle is smaller than200 μm. In other embodiments, the microparticle is about 20 nm. In someembodiments, the microparticle is from about 0.01 μm to about 200 μm,from about 0.02 μm to about 200 μm, from about 0.05 μm to about 200 μm,from about 0.1 μm to about 200 μm, from about 0.5 μm to about 200 μm,from about 1 μm to about 200 μm, from about 2 μm to about 200 μm, fromabout 5 μm to about 200 μm, from about 10 μm to about 200 μm, from about15 μm to about 200 μm, from about 20 μm to about 200 μm, from about 25μm to about 200 μm, from about 30 μm to about 200 μm, from about 35 μmto about 200 μm, from about 40 μm to about 200 μm, from about 45 μm toabout 200 μm, from about 50 μm to about 200 μm, from about 55 μm toabout 200 μm, from about 60 μm to about 200 μm, from about 65 μm toabout 200 μm, from about 70 μm to about 200 μm, from about 75 μm toabout 200 μm, from about 80 μm to about 200 μm, from about 85 μm toabout 200 μm, from about 90 μm to about 200 μm, from about 95 μm toabout 200 μm, from about 100 μm to about 200 μm, from about 101 μm toabout 200 μm, from about 102 μm to about 200 μm, from about 105 μm toabout 200 μm, from about 10 μm to about 200 μm, from about 115 μm toabout 200 μm, from about 120 μm to about 200 μm, from about 125 μm toabout 200 μm, from about 130 μm to about 200 μm, from about 135 μm toabout 200 μm, from about 140 μm to about 200 μm, from about 145 μm toabout 200 μm, from about 150 μm to about 200 μm, from about 155 μm toabout 200 μm, from about 160 μm to about 200 μm, from about 165 μm toabout 200 μm, from about 170 μm to about 200 μm, from about 175 μm toabout 200 μm, from about 180 μm to about 200 μm, from about 185 μm toabout 200 μm, from about 190 μm to about 200 μm, or from about 195 μm toabout 200 μm.

In some embodiments, the microparticle is from about 0.01 μm to about100 μm, from about 0.02 μm to about 100 μm, from about 0.05 μm to about100 μm, from about 0.1 μm to about 100 μm, from about 0.5 μm to about100 μm, from about 1 μm to about 100 μm, from about 2 μm to about 100μm, from about 5 μm to about 100 μm, from about 10 μm to about 100 μm,from about 15 μm to about 100 μm, from about 20 μm to about 100 μm, fromabout 25 μm to about 100 μm, from about 30 μm to about 100 μm, fromabout 35 μm to about 100 μm, from about 40 μm to about 100 μm, fromabout 45 μm to about 100 μm, from about 50 μm to about 100 μm, fromabout 55 μm to about 100 μm, from about 60 μm to about 100 μm, fromabout 65 μm to about 100 μm, from about 70 μm to about 100 μm, fromabout 75 μm to about 100 μm, from about 80 μm to about 100 μm, fromabout 85 μm to about 100 μm, from about 90 μm to about 100 μm, or fromabout 95 μm to about 100 μm.

In some embodiments, the microparticle is from about 0.01 μm to about 50μm, from about 0.02 μm to about 50 μm, from about 0.05 μm to about 50μm, from about 0.1 μm to about 50 μm, from about 0.5 μm to about 50 μm,from about 1 μm to about 50 μm, from about 2 μm to about 50 μm, fromabout 5 μm to about 50 μm, from about 10 μm to about 50 μm, from about15 μm to about 50 μm, from about 20 μm to about 50 μm, from about 25 μmto about 50 μm, from about 30 μm to about 50 μm, from about 35 μm toabout 50 μm, from about 40 μm to about 50 μm, or from about 45 μm toabout 50 μm.

In some embodiments, the microparticle is from about 0.01 μm to about 20μm, from about 0.02 μm to about 20 μm, from about 0.05 μm to about 20μm, from about 0.1 μm to about 20 μm, from about 0.5 μm to about 20 μm,from about 1 μm to about 20 μm, from about 2 μm to about 20 μm, fromabout 5 μm to about 20 μm, from about 10 μm to about 20 μm, or fromabout 15 μm to about 20 μm.

In some embodiments, the microparticle is from about 0.01 μm to about 10μm, from about 0.02 μm to about 10 μm, from about 0.05 μm to about 10μm, from about 0.1 μm to about 10 μm, from about 0.2 μm to about 10 μm,from about 0.3 μm to about 10 μm, from about 0.4 μm to about 10 μm, fromabout 0.5 μm to about 10 μm, from about 0.6 μm to about 10 μm, fromabout 0.7 μm to about 10 μm, from about 0.8 μm to about 10 μm, fromabout 0.9 μm to about 10 μm, from about 1 μm to about 10 μm, from about2 μm to about 10 μm or from about 5 μm to about 10 μm.

In embodiments, the microparticle is about 0.9 μm. In embodiments, themicroparticle has a diameter of about 0.9 μm. In embodiments, themicroparticle is about 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, 10, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 μm. In otherembodiments, the microparticle has a diameter of about 0.01, 0.02, 0.05,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, or 200 μm. The numerical values above represent the size of themicroparticle in μm.

In embodiments, the microparticle is a functionalized microbead. Wherethe microparticle is a functionalized microbead, the microparticle mayinclude any reactive moiety suitable for the conjugate chemistriesdescribed herein. The term “functionalized” as provided herein refers toa compound or domain (e.g., microparticle, linker, ligand domain,nucleic acid domain, nucleic acid sequence) including a reactive moietyor reactive functional groups used for conjugate chemistries asdescribed herein. For example, a functionalized microbead may includeone or more reactive moieties, such as any of an amino, carboxyl, thiol,or hydroxyl reactive moiety, incorporated on its surface. Inembodiments, a first functionalized group allows for attachment of theligand domain through a first linker. In embodiments, a secondfunctionalized group allows for attachment of the nucleic acid domainthrough a second linker. In embodiments, the first and the secondfunctionalized group are independently different. Therefore, the liganddomain may be attached to the microparticle through a first linker byconjugation to a different functionalized group than the nucleic aciddomain. In embodiments, a third functionalized group connects themicroparticle to a solid support. Therefore, in embodiments themicroparticle is covalently attached to a solid support.

The microparticle provided herein may include a polymer. In such a casethe polymers will carry the reactive moieties to be activated. Thepolymer may be selected from any suitable class of compounds, forexample, polyethylene glycols, polyethylene imides, polysaccharides,polypeptides, or polynucleotides. In embodiments, the microparticleincludes bis-amino polyethyleneglycol 3000 and hydroxyl-polyethyleneglycol 3000. In embodiments, the microparticle includes a polymer layer.Attachment of the polymers to the microparticle may be effected by avariety of methods which are readily apparent to a person skilled in theart. For example, polymers bearing trichlorosilyl or trisalkoxy groupsmay be reacted with hydroxyl groups on the microparticle to formsiloxane bonds. Attachment to a gold or silver microparticle may takeplace via thiol groups on the polymer. Alternatively, the polymer may beattached via an intermediate species, such as a self-assembled monolayerof alkanethiols. The type of polymers selected, and the method selectedfor attaching the polymers to the microparticle, will thus depend on thepolymer having suitable reactivity for being attached to themicroparticle surface, and on the properties of the polymers regardingnon-specific adsorption to, especially, DNA or peptides. The reactivemoieties may be present on the polymer or may be added to the polymer bythe addition of single or multiple reactive moieties. Optionally, aspacer arm (e.g., linker) can be used to provide flexibility to thebinding nucleic acid domain or ligand domain allowing it to interactwith its environment in a way which minimizes steric hindrance with themicroparticle.

In embodiments, the functionalized microbead is a magnetic polymer-based(polymeric) microbead. In embodiments, the microbead is a ProMag™microsphere. In embodiments, the microbead includes more than onepolymer. In embodiments, the microbead includes a first polymer and asecond polymer, wherein the first polymer and the second polymer arechemically different. In embodiments, the first polymer is bis-aminopolyethyleneglycol 3000 and the second polymer is hydroxyl-polyethyleneglycol 3000. In embodiments, the first polymer includes a first reactivemoiety and the second polymer includes a second reactive moiety. Areactive moiety as referred to herein includes any of the functionalmoieties useful for conjugate chemistry as described herein. Inembodiments, the first reactive moiety is an amino functional group andthe second reactive moiety is a hydroxyl functional group. Inembodiments, the hydroxyl functional group is reacted to form anazidoactate moiety. In embodiments, the first reactive moiety (e.g.,amino functional group) is reacted with a reactive moiety (e.g., acarboxyl functional group) of the first linker. In embodiments, theazidoacetate moiety is reacted with a reactive moiety (e.g., an alkynylfunctional group) of the second linker.

In embodiments, the microparticle is a polymeric microbead. Inembodiments, the microparticle is a dendrimer. A “dendrimer” as referredto herein is a spherical polymeric molecule made from two monomers(e.g., acrylic acid and a diamine). Dendrimers are precisely definedchemical structures that consist of a series of chemical shells built ona small core molecule. Each shell consists of two chemicals, always inthe same order. In embodiments, the microparticle is a branched polymer.In embodiments, the microparticle is a magnetic polymeric microbead. Inembodiments, the microparticle is a carboxymethyldextran functionalizedmicrobead. In embodiments, the microparticle is a polytheylene glycolfunctionalized microbead. In further embodiments, the polytheyleneglycol functionalized microbead includes orthogonally protected amines.In embodiments, the microparticle is a magnetic microbead. Inembodiments, the microparticle is a metallic microbead. In embodiments,the microparticle is a silica microbead.

As depicted in FIG. 1 the microparticles provided herein includingembodiments thereof may include a plurality of attachment points for theattachment of a plurality of a first, second and third linker. Themicroparticle may include a plurality of first attachment points for thefirst linker attaching the ligand domain, a plurality of secondattachment points for the second linker attaching the nucleic aciddomain and a plurality of third attachment points for the third linkerattaching the microparticle to a solid support. The total number ofattachment points per microparticle may be about 25-50 attomoles. Wherethe total number of attachment points corresponds to 100%, the number offirst attachment points may be more than about 1% and less than about20%. Where the total number of attachment points corresponds to 100%,the number of second attachment points may be more than about 40% andless than about 90%. Where the total number of attachment pointscorresponds to 100%, the number of third attachment points may be morethan about 0% and less than about 50%.

A “ligand domain” as provided herein is a domain capable of binding aligand binder (e.g., analyte, biomolecule). In embodiments, the liganddomain is a peptide. In embodiments, the ligand domain is a polypeptide.In embodiments, the ligand domain includes a surface glycoprotein orfragments thereof. In embodiments, the ligand domain has a proteinsequence corresponding to amino acid position 98-106 of Human influenzahemagglutinin (HA) protein. In embodiments, the ligand domain includesthe sequence of SEQ ID NO:17 or SEQ ID NO:18.

In embodiments, the ligand domain includes a protecting moiety attachedto a reactive moiety (e.g., a carboxyl functional group) of the liganddomain. As used herein, a protecting moiety is a chemical moietycovalently attached to a ligand domain that prevents the ligand domainfrom binding a ligand binder, wherein the protecting moiety may beremoved, for example, by chemical means when desired. In embodiments,the protecting moiety is fluorenylmethyloxycarbonyl. In embodiments, theprotecting moiety is tert-butyl or carboxybenzyl. Where the liganddomain includes a protecting moiety the ligand domain may be a sidechain protected polyamide. Where the ligand domain includes a protectingmoiety it may also be referred to herein as “synthetic intermediate” or“synthetic precursor.” In embodiments, the protecting moiety includes anamino acid side chain. In embodiments, the protecting moiety includes anamino terminus (e.g. a terminal —NH₂ group) or a carboxy terminus (e.g.a terminal —COOH group). In embodiments, the protecting moiety isattached to an amino acid side chain. In embodiments, the protectingmoiety is attached to an amino terminus (e.g. a terminal —NH₂ group) ora carboxy terminus (e.g. a terminal —COOH group). In the presence of theprotecting moiety the ligand domain is not capable of binding a ligandbinder. Thus, in embodiments the ligand domain includes a protectingmoiety and is not bound to a ligand binder. Upon removal of theprotecting moiety and reacting the reactive moiety with a ligand domaincapable of binding a ligand binder is formed.

The ligand domain provided herein may be formed using any multi-stepsupport-bound synthesis compatible with the composition of themicroparticle and with the synthesis chemistry of the nucleic aciddomain provided herein. The ligand domain and the nucleic acid domainmay be simultaneously synthesized on the microparticle. Alternatively,the nucleic acid domain is synthesized on the microparticle after thesynthesis of the ligand domain. In embodiments, the attachment of theligand domain through the first linker is performed prior to theattachment of the nucleic domain through the second linker. Inembodiments, the attachment of the ligand domain through the firstlinker is performed simultaneously with the attachment of the nucleicdomain through the second linker. In embodiments, the ligand domain is apeptide. In embodiments, the ligand domain is a small molecule. Inembodiments, the ligand domain is a protein. In embodiments, the liganddomain binds to a ligand binder. The ligand domain may be attached to adetectable moiety (e.g., a fluorescent moiety, luminescent moiety,colorimetric moiety, phosphorescent moiety, radioactive moiety orelectroactive moiety).

A “ligand binder” as used herein refers to an agent (e.g., atom,molecule, ion, molecular ion, compound or particle) capable of binding aligand domain provided herein including embodiments thereof. Ligandbinders include without limitation, biomolecules (e.g., hormones,cytokines, proteins, nucleic acids, lipids, carbohydrates, cellularmembrane antigens and receptors (neural, hormonal, nutrient, and cellsurface receptors or their ligands); whole cells or lysates thereof(e.g., prokaryotic (e.g., pathogenic bacteria), eukaryotic cells (e.g.,mammalian tumor cells); viruses (e.g., retroviruses, herpesviruses,adenoviruses, lentiviruses); and spores); chemicals (e.g., solvents,polymers, organic materials); therapeutic molecules (e.g., therapeuticdrugs, abused drugs, antibiotics); or environmental pollutants (e.g.,pesticides, insecticides, toxins). The ligand binder may be a protein, amixture of proteins, a nucleic acid, a mixture of nucleic acids, a smallmolecule, a mixture of small molecules, an element, a mixture ofelements, a synthetic polymer, a mixture of synthetic polymers, celllysate. In embodiments, the ligand binder is a biomolecule. Inembodiments, the biomolecule is a nucleic acid. In embodiments, thebiomolecule is a protein (e.g. antibody). In embodiments, the ligandbinder is an antibody. In embodiments, the ligand binder is an anti-HAantibody. In embodiments, the ligand binder is an anti-Myc antibody. Inembodiments, the ligand domain binds a peptide of SEQ ID NO:17. Inembodiments, the ligand domain binds a peptide of SEQ ID NO:18. Inembodiments, the ligand binder is attached to a detectable moiety. Inembodiments, the detectable moiety is a fluorescent moiety. Inembodiments, the ligand binder is a small molecule. In embodiments, theligand domain is not bound to a ligand binder. Where the ligand domainis not bound to a ligand binder, the ligand domain may include aprotecting moiety or any other applicable modification rendering theligand domain inert. The ligand binder may be attached to a detectablemoiety (e.g., a fluorescent moiety, luminescent moiety, colorimetricmoiety, phosphorescent moiety, radioactive moiety or electroactivemoiety).

As described above a “nucleic acid domain” as provided herein includes anucleic acid sequence corresponding to the individual building blocks ofthe ligand domain and the order in which these building blocks areincorporated in said ligand domain. Therefore, each microparticleincludes a unique ligand domain and a corresponding nucleic acid domainencoding specific nucleic acid sequences corresponding to the buildingblocks of the ligand domain and the order in which they wereincorporated in the ligand domain. The nucleic acid domains providedherein are also referred to as “tag” or “encoding tag.” In embodiments,the nucleic acid domain includes a nucleic acid sequence. Inembodiments, the nucleic acid sequence is about 18 base pairs in length.In embodiments, the nucleic acid sequence is about 20 base pairs inlength. In embodiments, the nucleic acid sequence is 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,79, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 base pairs in length. Inembodiments, the nucleic acid sequence does not include cytosine.

In embodiments, the nucleic acid sequence includes a covalent linker. Inembodiments, the covalent linker connects two nucleic acid sequenceswithin a nucleic acid domain. In embodiments, the nucleic acid domainincludes at least two nucleic acid sequences connected through acovalent linker. In embodiments, the nucleic acid domain includes atleast four nucleic acid sequences connected through covalent linkers.Thus, in embodiments, the nucleic acid domain includes a first nucleicacid sequence, a second nucleic acid sequence, a third nucleic acidsequence and a forth nucleic acid sequence, wherein the first nucleicacid sequence is connected to the second nucleic acid sequence through afirst covalent linker, the second nucleic acid sequence is connected tothe third nucleic acid sequence through a second covalent linker and thethird nucleic acid sequence is connected to the forth nucleic acidsequence through a third covalent linker. In embodiments, the covalentlinker (e.g., first, second, third covalent linker) is a bond, —S(O)—,—S(O)₂NH—, —NHS(O)₂—, —C(O)O—, —OC(O)—, —C(O), —C(O)NH, —NH—, —NHC(O)—,—O—, —S—, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene. In embodiments, the covalent linker is a 1,3 triazolenelinker. In embodiments, the covalent linker has the structure:

In embodiments, the covalent linker includes the structure:

In formula (I), the point of attachment marked by * indicates theattachment of the covalent linker to a first nucleic acid sequence andthe attachment marked by ** indicates attachment point of the covalentlinker to a second nucleic acid sequence.

In embodiments, the nucleic acid domain includes a functionalizednucleic acid sequence. A functionalize nucleic acid as provided hereinincludes reactive functional groups used for conjugate chemistries asdescribed herein. In embodiments, the nucleic acid domain includes aplurality of functionalized nucleic acid sequences. Where the nucleicacid domain includes a plurality of functionalized nucleic acidsequences, the functionalized nucleic acid sequences are connectedthrough a plurality of covalent linkers. In embodiments, each of theplurality of covalent linkers is chemically different. Schematicillustrations of the synthesis of the nucleic domain and ligand domainon a microparticle are depicted in FIGS. 4, 5, and 6. As depicted inFIGS. 5 and 6 the nucleic acid domains attached to a microparticle maybe independently different depending on the synthesis used and mayinclude two or more nucleic acid sequences connected through a covalentlinker.

The nucleic acid domains provided herein including embodiments thereofare compatible with (i) the multi-step support-bound synthesis methodsapplied to form a ligand domain as provided herein, (ii) the compositionof the microparticle and (iii) the decoding procedures provided herein(i.e., identifying the composition of the ligand domain and its locationon an array). Useful decoding procedures include without limitationsequencing by hybridization or enzymatic-based sequencing procedures(e.g., sequencing by synthesis, sequencing by ligation). Thus, inembodiments, the nucleic acid sequence is bound to a complementarynucleic acid sequence. In embodiments, the complementary nucleic acidsequence includes a detectable moiety. In embodiments, the detectablemoiety is a fluorescent moiety. Upon hybridization of a complementarynucleic acid to said nucleic acid domain, the composition of the liganddomain and its location on the array can be determined. After theidentity of the ligand domain and its location on the array have beendetermined the nucleic acid domain may be removed (e.g., throughcleavage of the second linker), the ligand domain may be furthermodified (e.g., through reacting a reactive moiety of the ligand domain)and contacted with a ligand binder (e.g., biomolecule).

The linkers provided herein chemically link the microparticle and theligand domain (first linker), the microparticle and the nucleic aciddomain (second linker) or the microparticle and the solid support (thirdlinker). As described above the nucleic acid domain provided hereinincluding embodiments thereof may include two or more nucleic acidsequences connected through covalent linkers (e.g., a 1,3 triazolenelinker). Thus, in embodiments, the nucleic acid domain includes two ormore 1,3 triazolene linkers. The linkers provided herein (e.g., firstlinker, second linker, third linker) may be covalently attached to themicroparticle applying methods well known in the art and compatible withthe composition of the linker and the microparticle. The linkersprovided herein may include the conjugated product of reactive moietiesat the point of attachment to the microparticle, at the point ofattachment to the ligand domain, at the point of attachment to thenucleic acid domain, or at the point of attachment to the solid support.Thus, the linkers provided herein may be polyvalent and may be formed byconjugate chemistry techniques. Non-limiting examples of linkers usefulfor the compositions and methods provided herein (e.g., first linker,second linker, third linker) include alkyl groups (including substitutedalkyl groups and alkyl groups containing heteroatom moieties), withshort alkyl groups, esters, amide, amine, epoxy groups and ethyleneglycol or derivatives thereof. The linkers provided herein (e.g., firstlinker, second linker, third linker) may include a sulfone group,forming sulfonamide, an ester group or an ether group (e.g., triethylether).

In embodiments, the first linker is a bond, —S(O)—, —S(O)₂NH—,—NHS(O)₂—, —C(O)O—, —OC(O)—C(O)—, —C(O)NH—, —NH—, —NHC(O)—, —O—, —S—,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene. In embodiments,the second linker is a bond, —S(O)—, —S(O)₂NH—, —NHS(O)₂—, —C(O)O—,—OC(O)—C(O)—, —C(O)NH—, —NH—, —NHC(O)—, —O—, —S—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. In embodiments, the thirdlinker is a bond, —S(O)—, —S(O)₂NH—, —NHS(O)₂—, —C(O)O—, —OC(O)—C(O)—,—C(O)NH—, —NH—, —NHC(O)—, —O—, —S—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. In embodiments, the firstlinker includes the structure —N(H)—C(O)—. In embodiments, the firstlinker includes the structure —N(H)—C(O)—. Where the first linker hasthe structure —N(H)—C(O)—, the nitrogen is attached to thefunctionalized solid support (e.g., functionalize with bis-amino PEG3000) and the carbon is attached to the ligand domain. As describedabove, after the identity of the ligand domain and its location on thearray have been determined, the nucleic acid domain may be removedthrough cleavage of the second linker. In embodiments, the second linkeris a photocleavable linker. In embodiments, the second linker is an acidlabile linker. In embodiments, the second linker includes an ester. Inembodiments, the second linker is an alkali labile linker. Inembodiments, the second linker has the structure:

In embodiments, the second linker includes the structure:

In formula (II), the point of attachment marked by * indicates the pointof attachment to the functionalized solid support (e.g., functionalizedwith hydroxyl-amine PEG 3000) and the point of attachment marked by **indicates the point of attachment to the nucleic acid domain. Inembodiments, the second linker has the structure:

In formula (IIA), X is an integer from 20-300. In embodiments, x is 68.In formula (IIA), the point of attachment marked by * indicates thepoint of attachment to the solid support and the point of attachmentmarked by ** indicates the point of attachment to the nucleic aciddomain. In embodiments, the second linker has the structure

In formula (IIB), L¹ is a bond, —S(O)—, —S(O)₂NH—, —NHS(O)₂—, —C(O)O—,—OC(O)—C(O)—, —C(O)NH—, —NH—, —NHC(O)—, —O—, —S—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. In formula (IIB), the pointof attachment marked by * indicates the point of attachment to the solidsupport and the point of attachment marked by ** indicates the point ofattachment to the nucleic acid domain.

According to the embodiments provided herein the microparticles providedherein may include a plurality of ligand domains and a plurality ofnucleic acid domains attached through a plurality of first linkers and aplurality of second linkers, respectively (see FIG. 3, 4, or 5). Thus,in embodiments, the ligand domain is a plurality of ligand domainsattached through a plurality of first linkers. In embodiments, thenucleic acid domain is a plurality of nucleic acid domains attachedthrough a plurality of second linkers. In embodiments, the plurality ofnucleic acid domains attached to a single microparticle may be the sameor independently different (see FIG. 5 or 6).

The microparticles provided herein including embodiments thereof may beattached to a solid support. In embodiments, the solid support is aplanar support. In embodiments, the microparticle is connected through athird linker to the solid support. In embodiments, the microparticle isnon-covalently attached to the solid support (e.g. through electrostaticinteractions (e.g. ionic bond, hydrogen bond, halogen bond), van derWaals interactions (e.g. dipole-dipole, dipole-induced dipole, Londondispersion), ring stacking (pi effects), hydrophobic interactions andthe like). In embodiments, the microparticle is mechanically attached tothe solid support. Where a microparticle is mechanically attached to thesolid support it is physically held in place on the support throughmechanical means (e.g., a well). In embodiments, a plurality ofmicroparticles are covalently attached to the solid support. Inembodiments, the microparticle is attached to the solid support throughan amide linker. Thus, in embodiments, the third linker has thestructure —N(H)—C(O)—. In embodiments, the solid support includescarboxymethyldextran. In embodiments, the solid support includescarboxymethyldextran functionalized glass. In embodiments, the solidsupport is a silicon wafer.

In embodiments, the plurality of microparticles form a disordered array.A “disordered array” as referred to herein is an array ofmicroparticles, wherein the microparticles are randomly assembled on orattached to a solid support and do not form an ordered two- orthree-dimensional structure. In embodiments, the plurality ofmicroparticles form an ordered array. In an ordered array, themicroparticles are assembled on or attached to a solid support accordingto an two- or three-dimensional order. For example, a hexagonal arrayconsists of a plurality of microparticles assembled on or attached to asolid support such that each microparticle forms part of a hexagon,wherein each microparticle occupies one angle of the hexagon, andwherein the center of the hexagon is occupied by a seventhmicroparticle. In embodiments, the plurality of microparticles form anhexagonal array. In embodiments, the plurality of microparticles form asquare packed array. A square packed array consists of a plurality ofmicroparticles assembled on or attached to a solid support such thateach microparticle forms part of a square or rectangle consisting of atleast four microparticles. The formation of arrays is a method wellknown and used in the art and is described, inter alia, in U.S. Pat.Nos. 6,110,426; 7,615,368; 7,932,213; 6,824,987; 5,143,854; 8,795,967and Hughes T R et al. (2001) Nat. Biotech. 4, 342-347, which are herebyincorporated in their entirety and for all purposes. In embodiments, atleast about 10⁶ of the microparticles are attached to the solid support.In embodiments, each of the microparticles is different. In embodiments,about 10⁶ to 10⁹ of the microparticles are attached to the solidsupport. In embodiments, about 10⁹ of the microparticles are attached tothe solid support. In embodiments, about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰or 10¹¹ of the microparticles are attached to the solid support. Inembodiments, the array includes 10⁶ microparticles per squaremillimeter.

In embodiments, the array includes at least about 10,000 microparticlesper square millimeter. In embodiments, the array includes at least about20,000 microparticles per square millimeter. In embodiments, the arrayincludes at least about 30,000 microparticles per square millimeter. Inembodiments, the array includes at least about 40,000 microparticles persquare millimeter. In embodiments, the array includes at least about50,000 microparticles per square millimeter. In embodiments, the arrayincludes at least about 60,000 microparticles per square millimeter. Inembodiments, the array includes at least about 70,000 microparticles persquare millimeter. In embodiments, the array includes at least about80,000 microparticles per square millimeter. In embodiments, the arrayincludes at least about 90,000 microparticles per square millimeter. Inembodiments, the array includes at least about 100,000 microparticlesper square millimeter. In embodiments, the array includes at least about200,000 microparticles per square millimeter. In embodiments, the arrayincludes at least about 300,000 microparticles per square millimeter. Inembodiments, the array includes at least about 400,000 microparticlesper square millimeter. In embodiments, the array includes at least about500,000 microparticles per square millimeter. In embodiments, the arrayincludes at least about 600,000 microparticles per square millimeter. Inembodiments, the array includes at least about 700,000 microparticlesper square millimeter. In embodiments, the array includes at least about800,000 microparticles per square millimeter. In embodiments, the arrayincludes at least about 900,000 microparticles per square millimeter.

In embodiments, the array includes about 200,000 microparticles persquare millimeter. In embodiments, the array includes about 789,000microparticles per square millimeter. In embodiments, the array includesabout 591,715 microparticles per square millimeter. In embodiments, thearray includes from about 200,000 to about 800,000 microparticles persquare millimeter.

In embodiments, the array includes about one microparticle per 4.99square microns. In embodiments, the array includes about onemicroparticle per 1.46 square microns. In embodiments, the arrayincludes about one microparticle per 1.69 square microns. Inembodiments, the array includes about one microparticle per squaremicron.

In embodiments, the solid support includes a plurality of wells each ofthe wells capturing one of the microparticle. In embodiments, thenucleic acid domain is a nucleic acid sequence as described herein. Inembodiments, the nucleic acid sequence is bound to a complementarynucleic acid sequence. In embodiments, the complementary nucleic acidsequence includes a detectable moiety. In embodiments, the detectablemoiety is a fluorescent moiety.

In another aspect, a solid support attached to a microparticle isprovided, wherein the microparticle is covalently attached to (i) aligand domain through a first linker; and (ii) a cleaved linker moiety.A “cleaved linker moiety” as provided herein is a monovalent chemicalmoiety formed through the cleavage of a second linker as provided hereinincluding embodiments thereof. In embodiments, the cleaved linker moietyis a remnant of a cleavable linker. In embodiments, the cleaved linkermoiety is a primary alcohol. In embodiments, the cleaved linker moietyis an amide. In embodiments, the ligand domain includes a protectingmoiety attached to a reacting group. In embodiments, the ligand domainis bound to a ligand binder. In embodiments, the ligand domain is aplurality of ligand domains attached through a plurality of firstlinkers. In embodiments, the cleaved linker moiety is a plurality ofcleaved linker moieties. In embodiments, the solid support is a planarsupport. In embodiments, the microparticle is non-covalently attached tothe solid support. In embodiments, the microparticle is connectedthrough a third linker to the solid support. In embodiments, themicroparticle is mechanically attached to the solid support. Inembodiments, a plurality of microparticles are attached to the solidsupport. In embodiments, the plurality of microparticles forms adisordered array. In embodiments, the plurality of microparticles formsan ordered array. In embodiments, the plurality of microparticles formsa hexagonal array. In embodiments, the plurality of microparticles formsa square packed array. In embodiments, at least about 200,000 of themicroparticles are attached per square millimeter of solid support andwherein each of the microparticles is different. In embodiments, atleast about 10⁶ of the microparticles are attached to the solid supportand wherein each of the microparticles is different. In embodiments, themicroparticles are attached to the solid support. In embodiments, about10⁹ of the microparticles are attached to the solid support. Inembodiments, the solid support is within a detection device. Inembodiments, the detection device detects the ligand binder bound to theligand domain and identifies a location of the bound ligand binder onthe solid support.

Methods

In another aspect, a method of forming a cleaved microparticle isprovided. The method includes (i) attaching a microparticle as providedherein including embodiments thereof to a solid support, thereby formingan immobilized microparticle. (ii) The second linker of the immobilizedmicroparticle is cleaved, thereby forming a cleaved microparticle. Inembodiments, the method includes prior to the cleaving of step (ii) andafter the attaching of step (i), binding a complementary nucleic acidsequence to the nucleic acid domain. In embodiments, the cleavingincludes contacting the immobilized microparticle with a cleaving agent.In embodiments, the cleaving agent is an acid. In embodiments, thecleaving agent is trifluoroacetic acid. In embodiments, the cleavingagent is an alkali agent. In embodiments, the cleaving agent is ammoniumhydroxide. In embodiments, the cleaving agent is ammonia. Inembodiments, the cleaving agent is methylamine. In embodiments, thecleaving agent is a mixture of ammonium hydroxide and methylamine. Inembodiments, the cleaving is performed at room temperature. Inembodiments, the cleaving agent is UV irradiation. In embodiments, thecleaving agent is light irradiation. In embodiments, the cleaving doesnot include cleaving the first linker.

In embodiments, the method includes after the cleaving of step (ii), astep (iii) of reacting a reactive moiety of the ligand domain, therebyforming a reactive ligand domain and (iv) binding a ligand binder to thereactive ligand domain. In embodiments, the method includes after thecleaving of step (ii), a step (iii) of binding a ligand binder to theligand domain. In embodiments, the step (ii) of cleaving includesbinding a ligand binder to the ligand domain Thus, the cleaving of thesecond linker may occur simultaneously with the binding of a ligandbinder to the ligand domain. Alternatively, the binding of a ligandbinder to the ligand domain may occur after the cleaving of the secondlinker. In embodiments, the binding of a ligand binder to the liganddomain includes reacting a reactive moiety of the ligand domain.

In another aspect, a method of detecting a ligand binder is provided.The method includes (i) attaching a microparticle as provided hereinincluding embodiments thereof to a solid support, thereby forming animmobilized microparticle. (ii) A complementary nucleic acid is bound tothe nucleic acid domain of the immobilized microparticle and a locationof the nucleic acid domain on the solid support is determined, therebyforming a decoded and mapped microparticle. (iii) The second linker ofthe decoded and mapped microparticle is cleaved, thereby forming amapped and cleaved microparticle. (iv) A ligand binder is bound to theligand domain of the mapped and cleaved microparticle; and (v) alocation of the bound ligand binder on the solid support is identified,thereby detecting the ligand binder. In embodiments, the cleaving ofstep (iii) and the binding of step (iv) occur simultaneously. Inembodiments, the binding of a ligand binder to the ligand domainincludes reacting a reactive moiety of the ligand domain.

In another aspect, a method of detecting a ligand binder is provided.The method includes (i) contacting a ligand binder with a microparticleas provided herein including embodiments thereof thereby forming a boundligand binder. (ii) A location of the bound ligand binder is identifiedon the solid support, thereby detecting the ligand binder.

EXAMPLES

Using split pool library synthesis Applicants were able to increase thenumber of compounds displayed by at least 1,000× over current methods.The compositions provided herein are a highly diverse collection ofmolecules immobilized in an extremely dense array on solid support. Toprepare Applicants' system, the assembled precursor library isimmobilized into a planar array. The entire immobilized array isdecoded, as each library member now occupies a permanent and discretespace in a planar array, the decoding converts what was a chemicallyencoded library into a spatially addressed library. The chemicalencoding units are removed and subsequent synthetic transformations areperformed across the immobilized library, completing the librarysynthesis. The library can then be screened to identify moleculesdemonstrating useful function.

When encoded split pool synthesis is combined with established methodsof high density bead immobilization and oligonucleotide sequencing, thesystem allows for screens of fully decoded split pool libraries on ascale that was not previously possible. By analogy to next generationsequencing technology, fully decoded screening sets of up to 108 to 1010in a microarray format should be achievable. Provided herein are“tagless” libraries for further chemistry. By decoding prior toscreening, oligonucleotide tags can be removed which accomplishes twothings: (i) additional chemistry that would be incompatible with theoligonucleotide tag can be done on the immobilized library (chemicalincompatibility is one of the stated challenges of encoded combinatorialchemistry); (ii) removal of the tags eliminates the potential of thetags interfering with the assays of interest. The encoding chemistryenables Applicants to harness the power of split-pool synthesis forchemical library generation, yet allows for libraries to be evaluated inrelatively information rich screens, rather than selections. Therelative performance of all library members in a given assay areevaluated, not just selected ‘hits’. Compounds that prove problematic inone or more assays can be easily identified and flagged, reducing thenumber of potential false positives in subsequent screens. Thisfacilitates the development of structure activity relationships.

The final bioactive compounds that are screened for activity are formedafter decoding and removal of the tags. What is immobilized and decodedare protected, synthetic intermediates. For the invention providedherein, at least one additional chemical step is performed afterdecoding and removal of the nucleic acid domain to finish preparing the“bioactive agents” (this could include: deprotection, macrocyclizationetc).

The potential benefits of encoding a split-pool library synthesis usingnucleic acids are well known as are the synthetic challenges.Applicants' specific encoding strategy allows for the creation ofscreenable DNA encoded libraries in fewer linear chemicaltransformations per step than previously demonstrated, with eachencoding step being independently decodable.

The microparticle also referred to herein as “core” could be adendrimer, a hyperbranched polymer, a functionalized silica particle, afunctionalized polymer particle. The core could be magnetic. The corecould range in size from 20 nm to 200 microns in diameter. The core isfunctionalized with a reactive moiety that allows attachment of buildingblocks to make the library molecules, a different reactive moiety thatallows attachment of DNA tags for encoding, and, in some cases, a thirddifferent reactive moiety which aids in covalent immobilization of thecore to a surface.

The multidentate core can be a 0.9 micron magnetic polymeric beadfunctionalized with a surface of polyethylene glycol terminated withorthogonally protected amines.

The synthetic precursor (ligand domain including a protecting moiety)could be any product of multi-step support-bound synthesis, with therestriction that any synthetic transformations used to create thesynthetic precursor, once attached to the core particle must becompatible with the core chemical structure and are eitherorthogonal/compatible with the described encoding chemistry or performedprior to incorporation of the first encoding tags. The syntheticprecursors may be side chain protected polyamides.

The Encoding tags (nucleic acid domains) consist of unique,pre-synthesized, functionalized nucleic acid derivatives covalentlylinked directly to the core particle through a cleavable linker orindirectly through other encoding tags. Tag sequences are of sufficientlength and composition and encode entire library. To enable decoding ofspecific encoded synthetic steps, independent of other encoded syntheticsteps. For example: decoding the tags used to encode for the second stepin a synthesis should be completely independent of the ability to decodetags used to encode the first or third steps in a synthesis.Unsuccessful incorporation of tags at step one adversely impactincorporation of tags at step two. An inability to “decode” step onewill affect the ability to “decode” step two for a given encoded librarymember. Applicants' approach may require longer nucleic acid sequencesbut provides a more robust encoding/decoding process. Tags arecompatible with the chosen method of decoding or sequencing. Forexample; if sequencing of tags is to be performed by a process ofsequential hybridizations, tags should be relatively isothermal to oneanother and contain sufficient sequence differences such that undesiredcross hybridization is minimized. If sequencing of tags is to be donethrough any of the enzymatic based sequencing by synthesis or sequencingby ligation approaches, tags may require common primer binding sites.

Tag chemical structures: (i) stable and unreactive to the synthetictransformations used to construct the precursor library; (ii) removablefrom the core particle after it is sequenced; (iii) compatible withmethod of decoding/sequencing. For example, sequencing by hybridizationcould be done with DNA, PNA, LNA, RNA, modified RNA, modified DNAanalogues or some combinations thereof. For any enzymatic basedsequencing approaches, DNA is preferred. The protected oligonucleotidestructures could be used to improve chemical orthogonality, whereinprotecting moieties on the tag are removed prior to decoding. (forexample, the exocyclic amines of the nucleobases may be protected priorto or immediately following tag incorporation).

Tag cleavage site structures: (i) tags may be directly attached to thecore particle or indirectly to the core through other tags, all coreparticle attachment points for tags ultimately contain a site ofcleavability. (ii) preferred cleavable structures, following cleavage,leave the core particle surface free of reactive moieties which mayinterfere with subsequent chemistry or assays (e.g., ester cleavage sitewhich leave a free alcohol; trityl ether cleavage site which leave afree alcohol).

Tag linking structures: the encoding tags may be 20-mer DNA basedoligonucleotides with zero C content and 25% G content. The tags differfrom one another by at least a six base pair mismatch. The tags arelinked to the support and or one another through 1,3 triazole linkagesall ultimately connected to the core particle through a cleavable esterbond. Any method of attachment and any surface used must be compatiblewith the decoding conditions and microarray assay conditions, as well astag removal conditions, and any synthetic reaction conditions applied tothe array. Applicants have demonstrated immobilization on patternedquartz, patterned silicon, and carboxymethyldextran functionalizedglass.

DNA tag removal: Conditions are dependent on structure at tag cleavagesite. With ester linkage, Applicants have used ammonium hydroxide andmethylamine Ligand deprotection and further synthetic modifications.Applicants have removed all side chain protecting moieties frompolyamide structures. Selective side chain de-protection followed bymacrolactamization will be performed. Incorporation offluorophore-quencher pairs or solvatochromic moieties will be performed.The compositions provided herein may be useful, inter alia, formicroarray screens, fluorescence, colorimetric, or electrochemicalreadouts binding assays in which fluorescently labeled proteins ofinterest are incubated over the array to identify binders has beendemonstrated.

In embodiments, the prototype chips are at 2.4 micron C-C (center tocenter) spacing in a hexagonal array. That results in 4.99 squaremicrons per bead (less than the 5.76 square microns per bead expectedbecause the beads are hexagonally packed), or 20 million beads persquare centimeter, or 200,000 beads per square millimeter, or ˜376million particles within the area equivalent of a standard 25 mm by 75mm microscope slide.

In embodiments, particles have been immobilized into a 1.3 micron C-Cspacing hexagonal array. That results in 1.46 square microns per bead,or 78.9 million beads per square centimeter, or 789,000 beads per squaremillimeter, or 1.28 billion beads within the area equivalent to a 25 mmby 75 mm microscope slide.

In embodiments, particles have been immobilized into a 1.3 micron C-Cspacing square packed array. That results in 1.69 square microns perbead, or 59 million beads per square centimeter, or 591,715 beads persquare millimeter, or 1.11 billion beads within the area equivalent to a25 mm by 75 mm microscope slide.

In embodiments, an array is hexagonally packed with a density of 1 beadper square micron (C-C spacing of ˜1.075 microns).

Materials and Methods

Reagents

Triethylamine (TEA), Diisopropylethylamine (DIPEA),Diisopropylcarbodiimide (DIC), dimethylaminopyridine (DMAP),dimethylformamide (DMF), dimethylsulfoxide (DMSO), Triton X-100 (TX100),azidoacetic acid (Aza),1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU),(7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate(PYAOP), Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), Copper(I)bromide dimethyl sulfide complex (CuBrDMS), Boc-Glycine, andFmoc-Glycine were purchased from Sigma-Aldrich and used as received. Allother Fmoc protected amino acids were purchased from Novabiochem orAdvanced Chemtech. Water used in all wash and reaction buffers wasobtained from a Millipore MilliQ purification system. Peg reagents usedfor the initial microparticle functionalization were purchased from RappPolymere or Quanta Biodesign. Promag Beads are provided by BangsLaboratories as a 2.6% w/v solution in water and have the followingcharacteristics: 880 nm average diameter. Composed of iron oxideembedded within a highly crosslinked polymer matrix. Surface of theparticles displays free carboxylic acid reactive moieties at 440 nmoleequivalents per milligram. Approximately 2 billion microparticles arecontained within one milligram of the stock particles. All nucleic acidtags and fluorescently labeled complement sequences were purchased fromIDT.

TABLE 1 Oligo  SEQ  sequence name IDT Sequence code ID NO: Tag A₀/5Hexynyl/AAC CAC ACA CAC ACA ACC /3AmMO/  1 Tag A₁/5Hexynyl/AAC CAC ACA CAC CAA ACC /3AmMO/  2 Tag B₂/5Hexynyl/ACG AAC ACA CAC GTA CGA /3AmMO/  3 Tag C₂/5Hexynyl/CAA GAC ACA CAC GTC AAG /3AmMO/  4 Tag D₂/5Hexynyl/CCT TAC ACA CAC GTC CTT /3AmMO/  5 Tag B₃/5Hexynyl/ACG AAO ACA CAC TGA CGA /3AmMO/  6 Tag C₃/5Hexynyl/CAA GAC ACA CAC TGC AAG /3AmMO/  7 Tag D₃/5Hexynyl/CCT TAC ACA CAC TGC CTT /3AmMO/  8 Anti-B₂-Cy3/5Cy3/TCG TAC GTG TGT GTT CGT  9 Anti-C₂-Cy3/5Cy3/CTT GAC GTG TGT GTC TTG 10 Anti-D₂-Cy3/5Cy3/AAG GAC GTG TGT GTA AGG 11 Anti-B₃-AF647/5Alex647N/TCG TCA GTG TGT GTT CGT 12 Anti-C₃-FITC/56-FAM/CTT GCA GTG TGT GTC TTG 13 Anti-D₃-FITC/56-FAM/AAG GCA GTG TGT GTA AGG 14 Anti-A₀-Cy3/5Cy3/GGT TGT GTG TGT GTG GTT 15 Anti-A₁-FITC/56-FAM/GGT TTG GTG TGT GTG GTT 16

Ligand Domain Sequences

SEQ ID NO: 17 YPYDVPDYA. (HA-tag) SEQ ID NO: 18 EQKLISEEDL (Myc-tag)

Additional Reagent Abbreviations

DITx MilliQ water containing 1% Triton X-100

DMSOTx DMSO containing 1% Triton X-100

PBT 100 mM phosphate buffer, pH 7.0 containing 1% Triton X-100

AMA 1:1 mix of ammonium hydroxide and 30% methylamine in ethanol

General Microparticle Handling Procedures

Similar to most solid phase synthetic procedures, a typical reactioninvolves 1) dispersing the microparticles in a reaction solution 2)adding additional solvents or reagents as needed for the reaction 3)providing occasional or constant agitation at some specified reactiontemperature for some period of time and 4) following the reaction, thereagents and soluble byproducts of the reaction are separated from themicroparticles through a series of washes. The separation and washingsteps for Applicants' microparticles consists of multiple rounds of 1)magnetically assisted pelleting of the microparticles and aspiration ofthe supernatant followed by 2) resuspension of the microparticle pelletin a suitable wash solution.

ProMag Microparticle Functionalization

Initial PEGylation:

Large-scale functionalization to create pegylated beads (with a 4.5:1ratio of hydroxyl groups to amine groups) are done in order to eliminateany batch-to-batch variations. 250 uL of stock ProMag beads are washedwith DMFTx (1 mL, ×3) and suspended in 150 uL DMFTx. Amino hydroxy PEG3000 (270 mg) and bis amine PEG 3000 (30 mg) are weighed into a 1.5 mLconical tube and melted in an oil bath at 65 C. The beads are added tothe melted PEG and mixed thoroughly/heated. Fifty five microliters ofDIPEA is added to the reaction mix and vortexed followed by the additionof solid PyAOP (160 mg). The reaction ran at 65 C for 45 minutes whileheating with an oil bath. The final concentrations in the reaction endup being (200 mM PEG, 600 mM PyAOP, 600 mM DIPEA). After 45 minutes theremaining carboxylic acids on the surface are capped with 2-methoxyethylamine (250 uL), let incubate at the same temperature for 10 minutes thenwash the beads with DMFTx (1 mL, ×3), then resuspend with 250 uL DMFTx.A 50 uL aliquot taken out and washed with MilliQ water (400 uL, ×3) andloaded on the burned off clean/tared pan, put in the oven at 95 C andthe following method was ran. Jump to 95 C, isotherm for 10 min, ramp 20C/min until 500 C, isotherm for 30 minutes. [Starting mass: 1.5940 mg.At 100 C 98.86%. At 500 C 31.30%] This corresponds to 25.4% addedfunctionality, comparison to the last large batch which was 24.3% addedfunctionalization (1.704 mg, 99.81% at 100 C and 32.08% at 500 C) ascompared to the first two Large batches prepared by the same protocolwhich had an added funct of 25.6%.

BOC Protection (×2)

The rest of the beads are Boc protected by washing into DMSOTx (1 mL,×3), suspending in 170 uL DMFTx, adding 14 mg Boc-Gly OH, followed by22.3 uL TEA, and 30.4 mg HATU, in that order. The reaction ran at 65 Cfor 30 min in an oil bath, after which the beads were washed with DMFTx(500 mL, ×3) and subject to the same conditions one more time. The beadswere then subject to AMA (200 uL, 65 C, 5 min) then washed into DMFTx atstock concentration.

DIC/DMAP with Long Chain Azide (×3)

Beads are resuspended in 400 uL DMFTx, to this is added 80 uL of 500 mMlong chain azido acid (11.66 mg/100 uL), followed by 6.7 uL DIC, then 10uL of DMAP (80 mg/ml stock in DMF). These coupling conditions wererepeated a total of three times—washing with DMFTx (200 uL, ×3) inbetween. The Boc group is then removed by treatment with TFA for 15minutes. This large batch is then washed into PBT (400 uL, ×3) andsuspend in 400 uL (×4 dilution from stock). Protocol for attaching azidoacetic acid in a alkali cleavable form (connected to the microparticlethrough an ester bond) is performed in a similar fashion.

Standard HATU Amino Acid Coupling Conditions

400 mM HATU, 400 mM FMOC'ed amino acid, 800 mM TEA in 10% DITx, 90%DMSOTx, 65 C, 30 minutes

Beads are washed into DITx (100 uL, ×3), and suspended in 10 uL DITx. Tothis is added the FMOC'ed amino acid in 80 uL DMSOTx (400 mM for a 200uL r×n volume), followed by TEA (10 uL, 101.19 g/mole, 0.726 g/mL), andlastly HATU (15 mg, 380.23 g/mole) is added last as a solid and the tubeis set on the red heat block set at 65 C for 30 minutes. Doublecouplings are done (without washing in between) with all amino acids toensure 100% conversion.

Standard HATU coupling of azide to the amine reactive moiety on thenucleic acid tags 400 mM HATU, 400 mM 2-azidoacetic acid, 800 mM TEA in10% DITx, 90% DMSOTx, 65 C, 30 minutes

Beads are washed into PBT (100 uL, ×3), and suspended in 10 uL DITx. Tothis is added DMSOTx (80 uL), followed by 2-azidoacetic acid (3 uL,101.06 g/mole, 1.35 g/mL), followed by TEA (22.3 uL, 101.19 g/mole,0.726 g/mL), and lastly HATU (15 mg, 380.23 g/mole) is added last as asolid and the tube is set on the red heat block set at 65 C for 30minutes. Double couplings are done (without washing in between) with allacids to ensure 100% conversion.

Copper Catalyzed Azide Alkyne (Huisgen) Cycloaddition Conditions(Encoding Step)

Catalyst stock solution preparation: Cu(I)Br DMS (205.58 g/mole) hadpreviously been weighed out in the glovebox in 20 mL scintillationvials. DMSO (that is kept in the glovebox) is added to the vial suchthat a concentration of 4.5 mg/mL (22 mM) is achieved. THPTA(tris(3-hydroxypropyltriazolylmethyl)amine) (434.5 g/mole) is weighedout outside of the hood and dissolved to a concentration of 2.8 mg/125uL (52 mM). The Cu (I) and the ligand solutions are combined in a 1:2v:v ration of Cu(I) to ligand giving the following concentrations: 35 mMTHPTA and 7.3 mM Cu(I)Br DMS in 50% DMSO and 50% MilliQ water.

The beads are washed into PBT (100 uL, ×3) and suspended in 13 uL PBT.To this is added 10 uL oligo tag (from 500 uM stock solution). This istransferred into the glovebox with a minimum of 4 pump/purge cycles. Twomicroliters of the catalyst stock solution is added and the PCR tube isset on the PCR block set to 60 C for 30 minutes, after which thereaction is quenched with 500 mM EDTA (outside of the glove box) byadded 100 uL EDTA and incubating for 3 minutes before washing with PBT(200 uL, ×5).

Standard FMOC Deprotection Conditions

100 uL 20% piperadine in DMFTx for 10 minutes at rt (×3).

Competitive Hybridization Conditions, Example Using for DistinguishingBetween Tags A0 and A1

Anti-A0-Cy3, and Anti-A1-FITC are purchased from IDT and diluted tostocks of 500 μM in MilliQ water. A hybridization solution that is to beapplied to bead samples is prepared as follows. 40 μL of formamide, 20μL of 20×SSPE buffer (Sigma), 10 μL of DITx, 20 μL of DI, 5 μL ofAnti-A0-Cy3 stock, and 5 μL of Anti-A1-FITC stock are mixed in a singlePCR tube, and stored in the dark till use.

Samples of beads (1.6 μg) displaying A0 or A1 on their respectivesurfaces were placed in separate PCR tube, pelleted, and the supernatantwas removed by vacuum aspiration. Each bead pellet was immediatelysuspended in 25 μL of hybridization solution. The bead slurry mixtureswere allowed to hybridize over the next 15 minutes at room temperature,away from light. After the indicated time period, the bead samples werepelleted, the hybridization solution was removed by vacuum aspiration.Beads were then washed 3× with 25 μL of PBT, and finally suspended in 25μL pf PBT. 5 μL of this bead sample is removed, placed in a 1356 wellplate and imaged at the microscopy core using a Zeiss Observer, 63×water objective, 1.6 optovar in the brightfeild, DsRed and EGFPchannels. An extra 5 μL sample of each bead type was loaded in aseparate well of the plate and used to set the exposure times for theDsRed and EGFP by the 2014 Zeiss ZenBlue software. The additional A0sample was used to set the DsRed and the additional A1 sample was usedto set the EGFP. After setting the exposure time the unexposed A0 and A1samples were imaged using the same, fixed exposure times.

EMBODIMENTS

Embodiment 1. A microparticle covalently attached to: (i) a liganddomain through a first linker; and (ii) a nucleic acid domain through asecond linker, wherein said second linker is cleavable and said firstlinker is not cleavable under a condition that said second linker iscleavable.

Embodiment 2. The microparticle of embodiment 1, wherein said liganddomain comprises a protecting moiety attached to a reactive moiety.

Embodiment 3. The microparticle of embodiment 2, wherein said protectingmoiety comprises an amino acid side chain.

Embodiment 4. The microparticle of embodiment 2, wherein said protectingmoiety comprises an amino terminus or a carboxy terminus.

Embodiment 5. The microparticle of embodiment 1, wherein saidmicroparticle is a microbead.

Embodiment 6. The microparticle of embodiment 1, wherein saidmicroparticle is a functionalized microbead.

Embodiment 7. The microparticle of embodiment 1, wherein saidmicroparticle is a magnetic microbead.

Embodiment 8. The microparticle of embodiment 1, wherein saidmicroparticle is a metallic microbead.

Embodiment 9. The microparticle of embodiment 1, wherein saidmicroparticle is a silica microbead.

Embodiment 10. The microparticle of embodiment 1, wherein saidmicroparticle is a polymeric microbead.

Embodiment 11. The microparticle of embodiment 1, wherein saidmicroparticle a dendrimer.

Embodiment 12. The microparticle of embodiment 1, wherein saidmicroparticle is a branched polymer.

Embodiment 13. The microparticle of any one of embodiments 1-12, whereinsaid second linker is a photocleavable linker.

Embodiment 14. The microparticle of any one of embodiments 1-12, whereinsaid second linker is an acid labile linker.

Embodiment 15. The microparticle of any one of embodiments 1-12, whereinsaid second linker is an alkali labile linker.

Embodiment 16. The microparticle of any one of embodiments 1-15, whereinsaid ligand domain is a peptide.

Embodiment 17. The microparticle of any one of embodiments 1-15, whereinsaid ligand domain is a small molecule.

Embodiment 18. The microparticle of any one of embodiments 1-15, whereinsaid ligand domain is a protein.

Embodiment 19. The microparticle of embodiment 16, wherein said liganddomain binds to a ligand binder.

Embodiment 20. The microparticle of embodiment 19, wherein said ligandbinder is a biomolecule.

Embodiment 21. The microparticle of embodiment 20, wherein saidbiomolecule is a nucleic acid.

Embodiment 22. The microparticle of embodiment 20, wherein saidbiomolecule is a protein.

Embodiment 23. The microparticle of any one of embodiments 1-16, whereinsaid ligand domain is not bound to a ligand binder.

Embodiment 24. The microparticle of any one of embodiments 1-23, whereinsaid ligand domain is a plurality of ligand domains attached through aplurality of first linkers.

Embodiment 25. The microparticle of any one of embodiments 1-24, whereinsaid nucleic acid domain is a plurality of nucleic acid domains attachedthrough a plurality of second linkers.

Embodiment 26. The microparticle of any one of embodiments 1-25, whereinsaid microparticle is attached to a solid support.

Embodiment 27. The microparticle of embodiment 26, wherein said solidsupport is a planar support.

Embodiment 28. The microparticle of embodiment 26 or 27, wherein saidmicroparticle is connected through a third linker to said solid support.

Embodiment 29. The microparticle of embodiment 26 or 27, wherein saidmicroparticle is non-covalently attached to said solid support.

Embodiment 30. The microparticle of embodiment 26 or 27, wherein saidmicroparticle is mechanically attached to said solid support.

Embodiment 31. The microparticle of any one of embodiments 26-30,wherein a plurality of microparticles are attached to said solidsupport.

Embodiment 32. The microparticle of embodiment 31, wherein saidplurality of microparticles form a disordered array.

Embodiment 33. The microparticle of embodiment 31, wherein saidplurality of microparticles form an ordered array.

Embodiment 34. The microparticle of embodiment 31, wherein said theplurality of microparticles form an hexagonal array.

Embodiment 35. The microparticle of embodiment 31, wherein said theplurality of microparticles form a square packed array.

Embodiment 36. The microparticle of any one of embodiments 32-35,wherein said array includes at least about 200,000 microparticles persquare millimeter.

Embodiment 37. The microparticle of any one of embodiments 32-35,wherein said array includes about 200,000 microparticles per squaremillimeter.

Embodiment 38. The microparticle of any one of embodiments 32-35,wherein said array includes 789,000 microparticles per squaremillimeter.

Embodiment 39. The microparticle of any one of embodiments 32-35,wherein said array includes 591,715 microparticles per squaremillimeter.

Embodiment 40. The microparticle of any one of embodiments 31-35,wherein at least about 10⁶ of said microparticles are attached to saidsolid support.

Embodiment 41. The microparticle of embodiment 40, wherein each of saidmicroparticles is different.

Embodiment 42. The microparticle of embodiment 40, wherein about 10⁶ to10⁹ of said microparticles are attached to said solid support.

Embodiment 43. The microparticle of embodiment 42, wherein about 10⁹ ofsaid microparticles are attached to said solid support.

Embodiment 44. The microparticle of any one of embodiments 31-43,wherein said solid support comprises a plurality of wells each of saidwells capturing one of said microparticle.

Embodiment 45. The microparticle of any one of embodiments 1-44, whereinsaid nucleic acid domain comprises a nucleic acid sequence.

Embodiment 46. The microparticle of embodiment 45, wherein said nucleicacid sequence is bound to a complementary nucleic acid sequence.

Embodiment 47. The microparticle of embodiment 46, wherein saidcomplementary nucleic acid sequence comprises a detectable moiety.

Embodiment 48. The microparticle of embodiment 47, wherein saiddetectable moiety is a fluorescent moiety.

Embodiment 49. A solid support attached to a microparticle, wherein saidmicroparticle is covalently attached to: (i) a ligand domain through afirst linker; and (ii) a cleaved linker moiety.

Embodiment 50. The microparticle of embodiment 49, wherein said liganddomain comprises a protecting moiety attached to a reacting group.

Embodiment 51. The microparticle of embodiment 49, wherein said liganddomain is bound to a ligand binder.

Embodiment 52. The microparticle of embodiment 49 or 51, wherein saidcleaved linker moiety is a remnant of a cleavable linker.

Embodiment 53. The microparticle of embodiment 52, wherein said liganddomain is a plurality of ligand domains attached through a plurality offirst linkers.

Embodiment 54. The microparticle of embodiment 53, wherein said cleavedlinker moiety is a plurality of cleaved linker moieties.

Embodiment 55. The microparticle of any one of embodiments 49-54,wherein said solid support is a planar support.

Embodiment 56. The microparticle of any one of embodiments 49-55,wherein said microparticle is non-covalently attached to said solidsupport.

Embodiment 57. The microparticle of any one of embodiments 49-55,wherein said microparticle is connected through a third linker to saidsolid support.

Embodiment 58. The microparticle of any one of embodiments 49-55,wherein said microparticle is mechanically attached to said solidsupport.

Embodiment 59. The microparticle of any one of embodiments 49-58,wherein a plurality of microparticles are attached to said solidsupport.

Embodiment 60. The microparticle of embodiment 59, wherein saidplurality of microparticles form a disordered array.

Embodiment 61. The microparticle of embodiment 59, wherein saidplurality of microparticles form an ordered array.

Embodiment 62. The microparticle of any one of embodiments 59-62,wherein at least about 10⁶ of said microparticles are attached to saidsolid support and wherein each of said microparticles is different.

Embodiment 63. The microparticle of embodiment 62, wherein about 10⁶ to10⁹ of said microparticles are attached to said solid support.

Embodiment 64. The microparticle of embodiment 63, wherein about 10⁹ ofsaid microparticles are attached to said solid support.

Embodiment 65. The microparticle of any one of embodiments 59-64,wherein said solid support is within a detection device.

Embodiment 66. The microparticle of embodiment 65, wherein saiddetection device detects said ligand binder bound to said ligand domainand identifies a location of said bound ligand binder on said solidsupport.

Embodiment 67. A method of forming a cleaved microparticle, said methodcomprising:

(i) attaching a microparticle of any one of embodiments 1-25 to a solidsupport, thereby forming an immobilized microparticle;

(ii) cleaving said second linker of said immobilized microparticle,thereby forming a cleaved microparticle.

Embodiment 68. The method of embodiment 67, further comprising prior tosaid cleaving of step (ii) and after said attaching of step (i), bindinga complementary nucleic acid sequence to said nucleic acid domain.

Embodiment 69. The method of embodiment 67 or 68, wherein said cleavingcomprises contacting said immobilized microparticle with a cleavingagent.

Embodiment 70. The method of embodiment 69, wherein said cleaving agentis an acid.

Embodiment 71. The method of embodiment 70, wherein said cleaving agentis trifluoroacetic acid.

Embodiment 72. The method of embodiment 67 or 68, wherein said cleavingdoes not comprise cleaving said first linker.

Embodiment 73. The method of embodiment 69, wherein said cleaving agentis an alkali agent.

Embodiment 74. A method of detecting a ligand binder, said methodcomprising: (i) attaching a microparticle of any one of embodiments 1-25to a solid support, thereby forming an immobilized microparticle; (ii)binding a complementary nucleic acid to said nucleic acid domain of saidimmobilized microparticle and determining a location of said nucleicacid domain on said solid support, thereby forming a decoded and mappedmicroparticle; (iii) cleaving said second linker of said decoded andmapped microparticle, thereby forming a mapped and cleavedmicroparticle; (iv) binding a ligand binder to said ligand domain ofsaid mapped and cleaved microparticle; and (v) identifying a location ofsaid bound ligand binder on said solid support, thereby detecting saidligand binder.

Embodiment 75. A method of detecting a ligand binder, said methodcomprising: (i) contacting a ligand binder with a microparticle of anyone of embodiments 49-58; thereby forming a bound ligand binder; and(ii) identifying a location of said bound ligand binder on said solidsupport, thereby detecting said ligand binder.

What is claimed is:
 1. A microparticle immobilized on solid support,wherein said microparticle is covalently attached to: (i) a liganddomain through a first linker; and (ii) a nucleic acid domain through asecond linker, wherein said nucleic acid domain identifies said liganddomain; wherein said ligand domain is selected from the group consistingof a peptide, a small molecule, and a protein; wherein said secondlinker comprises an acyl group, a sulfonamide group, or a photocleavablegroup; and wherein said nucleic acid domain is removable under acondition that said ligand domain is not removable.
 2. The microparticleof claim 1, wherein said ligand domain comprises a removable protectingmoiety covalently attached to a reactive moiety.
 3. The microparticle ofclaim 2, wherein said reactive moiety is selected from the groupconsisting of a carboxylic acid, an amine, a thiol, or an alcohol. 4.The microparticle of claim 2, wherein said removable protecting moietycomprises an amino acid side chain, an amino terminus, or a carboxyterminus.
 5. The microparticle of claim 1, wherein said microparticle isselected from the group consisting of a functionalized microbead, amagnetic microbead, a metallic microbead, a polymeric microbead, asilica microbead, a dendrimer, and a branched polymer.
 6. Themicroparticle of claim 1, wherein said peptide is selected from thegroup consisting of a macrocyclic peptide, an acyclic precursor to amacrocyclic peptide, a peptidomimetic, a polyamide, and a macrolactam.7. The microparticle of claim 1, wherein said peptide further comprisesnon-peptide functionality.
 8. The microparticle of claim 1, wherein saidligand domain is bound to a ligand binder.
 9. The microparticle of claim1, wherein said ligand domain comprises a plurality of ligand domainsattached through a plurality of first linkers.
 10. The microparticle ofclaim 1, wherein said nucleic acid domain comprises a plurality ofnucleic acid domains attached through a plurality of second linkers. 11.The microparticle of claim 1, wherein said nucleic acid sequence isbound to a detectably labeled complementary nucleic acid sequence.
 12. Ahigh density ordered array, said high density ordered array comprising asolid support comprising a plurality of wells, wherein each of saidplurality of wells further comprises an immobilized microparticle, andwherein each of said immobilized microparticle is covalently attachedto: (i) a ligand domain through a first linker; and (ii) a nucleic aciddomain through a second linker; wherein said nucleic acid domainidentifies said ligand domain; wherein said ligand domain is selectedfrom the group consisting of a peptide, a small molecule, and a protein;wherein said second linker comprises an acyl group, a sulfonamide group,or a photocleavable group; and wherein said nucleic acid domain isremovable under a condition that said ligand domain is not removable.13. The high density ordered array of claim 12, wherein said liganddomain comprises a removable protecting moiety attached to a reactivemoiety.
 14. The high density ordered array of claim 12, wherein saidligand domain is bound to a ligand binder.
 15. The high density orderedarray of claim 12, wherein said peptide is selected from the groupconsisting of a macrocyclic peptide, an acyclic precursor to amacrocyclic peptide, a peptidomimetic, a polyamide, and a macrolactam.16. The high density ordered array of claim 12, wherein said peptidefurther comprises non-peptide functionality.
 17. The high densityordered array of claim 12, wherein said high density ordered arrayincludes at least 10,000 microparticles per square millimeter.
 18. The shigh density ordered array of claim 17, wherein said high densityordered array includes at least 50,000 microparticles per squaremillimeter.
 19. The high density ordered array of claim 18, wherein saidhigh density ordered array includes at least 150,000 microparticles persquare millimeter.
 20. The high density ordered array of claim 19,wherein said high density ordered array includes at least 250,000microparticles per square millimeter.
 21. The high density ordered arrayof claim 12, wherein said high density ordered array comprises between10⁶ to 10¹¹ of said immobilized microparticles.
 22. The high densityordered array of claim 12, wherein said condition is selected from thegroup consisting of UV irradiation, light irradiation, trifluoroaceticacid, ammonium hydroxide, ammonia, methylamine, and a mixture ofammonium hydroxide and methylamine.
 23. The high density ordered arrayof claim 12, wherein said ordered array is a square packed array or ahexagonal array.
 24. The high density ordered array of claim 12, whereinsaid second linker is a photocleavable linker, an acid labile linker, oran alkali labile linker.
 25. The high density ordered array of claim 12,wherein said nucleic acid domain is bound to a detectably labeledcomplementary nucleic acid sequence.
 26. The high density ordered arrayof claim 12, wherein said high density ordered array is within adetection device, wherein said detection device detects said ligandbinder bound to said ligand domain and identifies a location of saidbound ligand binder on said solid support.